Glycosylated VEGF-B and method for increasing the amount of soluble VEGF-B

ABSTRACT

N-glycosylated VEGF-B proteins, nucleic molecule encoding these proteins, pharmaceutical compositions containing them and a method for increasing the amount of a soluble VEGF-B protein. The VEGF-B proteins are useful in stimulating and maintaining angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional PatentApplication No. 60/220,824, filed Jul. 26, 2000.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the discovery that N-glycosylation ofVEGF-B causes an increase in soluble proteins.

[0003] The two major components of the mammalian vascular system areendothelial cells and smooth muscle cells. The endothelial cells formthe lining of the inner surface of all blood vessels and lymphaticvessels in the mammal. The formation of new blood vessels can occur bytwo different processes, vasculogenesis or angiogenesis (for a reviewsee Risau, W., Nature 386:671-674 (1997)). Vasculogenesis ischaracterized by the in situ differentiation of endothelial cellprecursors to mature endothelial cells and association of these cells toform vessels, such as occurs in the formation of the primary vascularplexus in the early embryo. In contrast, angiogenesis, the formation ofblood vessels by growth and branching of pre-existing vessels, isimportant in later embryogenesis and is responsible for most of theblood vessel growth which occurs in the adult. Angiogenesis is aphysiologically complex process involving proliferation of endothelialcells, degradation of extracellular matrix, branching of vessels andsubsequent cell adhesion events. In the adult, angiogenesis is tightlycontrolled and limited under normal circumstances to the femalereproductive system. However angiogenesis can be switched on in responseto tissue damage. Also solid tumors are able to induce angiogenesis insurrounding tissue, thus sustaining tumor growth and facilitating theformation of metastases (Folkman, J., Nature Med. 1:27-31, (1995)). Themolecular mechanisms underlying the complex angiogenic processes are farfrom being understood.

[0004] Angiogenesis is also involved in a number of pathologicalconditions, where it plays a role or is involved directly in differentsequelae of the disease. Some examples include neovascularizationassociated with various liver diseases, neovascular sequelae ofdiabetes, neovascular sequelae to hypertension, neovascularization inpost trauma, neovascularization due to head trauma, neovascularizationin chronic liver infection (e.g. chronic hepatitis), neovascularizationdue to heat or cold trauma, dysfunction related to excess of hormone,creation of hemangiomas and restenosis following angioplasty. Inarthritis, new capillaries invade the joint and destroy cartilage. Indiabetes, new capillaries in the retina invade the vitreous humour,causing bleeding and blindness (Folkman, J. and Shing, Y., J. Biol.Chem. 267:10931-10934 (1992)). The role of angiogenic factors in theseand other diseases has not yet been clearly established.

[0005] Because of the crucial role of angiogenesis in so manyphysiological and pathological processes, factors involved in thecontrol of angiogenesis have been intensively investigated. A number ofgrowth factors have been shown to be involved in the regulation ofangiogenesis. These include fibroblast growth factors (FGFs),platelet-derived growth factors (PDGFs), transforming growth factoralpha (TGF), and hepatocyte growth factor (HGF). See for example Folkmanet al, J. Biol. Chem., 267:10931-10934 (1992) for a review.

[0006] It has been suggested that a particular family of endothelialcell-specific growth factors known as the vascular endothelial growthfactors (VEGFs) and their corresponding receptors are primarilyresponsible for stimulation of endothelial cell growth anddifferentiation, and for certain functions of the differentiated cells.These factors are members of the PDGF/VEGF family, and appear to actprimarily via endothelial receptor tyrosine kinases (RTKs). ThePDGF/VEGF family of growth factors belongs to the cystine-knotsuperfamily of growth factors, which also includes the neurotrophins andtransforming growth factor-β.

[0007] Eight different proteins have been identified in the PDGF/VEGFfamily, namely two PDGFs (A and B), VEGF and five members that areclosely related to VEGF. The five members closely related to VEGF are:VEGF-B, described in International Patent Application No. WO 96/26736and in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute forCancer Research and The University of Helsinki; VEGF-C or VEGF2,described in Joukov et al, EMBO J. 15:290-298 (1996), Lee et al, Proc.Natl. Acad. Sci. USA 93:1988-1992 (1996), and U.S. Pat. Nos. 5,932,540and 5,935,540 by Human Genome Sciences, Inc; VEGF-D, described inInternational Patent Application No. PCT/US97/14696 (WO 98/07832), andAchen et al, Proc. Natl. Acad. Sci. USA 95:548-553 (1998); the placentagrowth factor (PlGF), described in Maglione et al, Proc. Natl. Acad.Sci. USA 88:9267-9271 (1991); and VEGF3, described in InternationalPatent Application No. PCT/US95/07283 (WO 96/39421) by Human GenomeSciences, Inc. Each VEGF family member has between 30% and 45% aminoacid sequence identity with VEGF in their VEGF homology domain (VHD).This VEGF homology domain contains the eight conserved cysteine residueswhich form the cystine-knot motif. In their active, physiological state,the proteins are dimers. Functional characteristics of the VEGF familyinclude varying degrees of mitogenicity for endothelial cells andrelated cell types, induction of vascular permeability and angiogenicand lymphangiogenic properties.

[0008] Vascular endothelial growth factor (VEGF) is a homodimericglycoprotein that has been isolated from several sources. VEGF showshighly specific mitogenic activity for endothelial cells. VEGF hasimportant regulatory functions in the formation of new blood vesselsduring embryonic vasculogenesis and in angiogenesis during adult life(Carmeliet et al., Nature, 380: 435-439, (1996); Ferrara et al., Nature,380: 439-442, (1996); reviewed in Ferrara and Davis-Smyth, EndocrineRev., 18: 4-25, (1997)). The significance of the role played by VEGF hasbeen demonstrated in studies showing that inactivation of a single VEGFallele results in embryonic lethality due to failed development of thevasculature (Carmeliet et al., Nature, 380: 435-439, (1996); Ferrara etal., Nature, 380: 439-442, (1996)). The isolation and properties of VEGFhave been reviewed; see Ferrara et al., J. Cellular Biochem., 47:211-218, (1991) and Connolly, J. Cellular Biochem., 47: 219-223, (1991).

[0009] In addition, VEGF has strong chemoattractant activity towardsmonocytes, can induce the plasminogen activator and the plasminogenactivator inhibitor in endothelial cells, and can also inducemicrovascular permeability. Because of the latter activity, it issometimes referred to as vascular permeability factor (VPF). VEGF isalso chemotactic for certain hematopoetic cells. Recent literatureindicates that VEGF blocks maturation of dendritic cells and therebyreduces the effectiveness of the immune response to tumors (many tumorssecrete VEGF) (Gabrilovich et al., Blood 92: 4150-4166, (1998);Gabrilovich et al., Clinical Cancer Research 5: 2963-2970, (1999)).

[0010] Vascular endothelial growth factor B (VEGF-B) has similarangiogenic and other properties to those of VEGF, but is distributed andexpressed in tissues differently from VEGF. In particular, VEGF-B isvery strongly expressed in heart, and only weakly in lung, whereas thereverse is the case for VEGF (olofsson, B. et al, Proc. Natl. Acad. Sci.USA 93:2576-2581 (1996)). RT-PCR assays have demonstrated the presenceof VEGF-B mRNA in melanoma, normal skin, and muscle. This suggests thatVEGF and VEGF-B, despite the fact that they are co-expressed in manytissues, may have functional differences. A comparison of the PDGF/VEGFfamily of growth factors reveals that the 167 amino acid isoform ofVEGF-B is the only family member that is completely devoid of anyglycosylation. Gene targeting studies have shown that VEGF-B deficiencyresults in mild cardiac phenotype, and impaired coronary vasculature(Bellomo et al, Circ. Res. 86:E29-35 (2000)).

[0011] Human VEGF-B was isolated using a yeast co-hybrid interactiontrap screening technique by screening for cellular proteins which mightinteract with cellular retinoic acid-binding protein type I (CRABP-I).The isolation and characteristics including nucleotide and amino acidsequences for both the human and mouse VEGF-B are described in detail inInternational Application No. WO 96/26736 and in U.S. Pat. Nos.5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and TheUniversity of Helsinki and in Olofsson et al, Proc. Natl. Acad. Sci. USA93:2576-2581 (1996). The nucleotide sequence for human VEGF-B is alsofound at GenBank Accession No. U48801. The entire disclosures of WO96/26736, U.S. Pat. No. 5,840,693 and 5,607,918 are incorporated hereinby reference. The mouse and human genes for VEGF-B are almost identical,and both span about 4 kb of DNA. The genes are composed of seven exonsand their exon-intron organization resembles that of the VEGF and PlGFgenes (Grimmond et al, Genome Res. 6:124-131 (1996); Olofsson et al, J.Biol. Chem. 271:19310-19317 (1996); Townson et al, Biochem. Biophys.Res. Commun. 220:922-928 (1996)).

[0012] VEGF-C was isolated from conditioned media of the PC-3 prostateadenocarcinoma cell line (CRL1435) by screening for ability of themedium to induce tyrosine phosphorylation of the endothelialcell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cellstransfected to express VEGFR-3. VEGF-C was purified using affinitychromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNAlibrary. Its isolation and characteristics are described in detail inJoukov et al., EMBO J., 15: 290-298, (1996).

[0013] VEGF-D was isolated from a human breast cDNA library,commercially available from Clontech, by screening with an expressedsequence tag obtained from a human cDNA library designated “SoaresBreast 3NbHBst” as a hybridization probe (Achen et al, Proc. Natl. Acad.Sci. USA, 95: 548-553, (1998)). Its isolation and characteristics aredescribed in detail in International Patent Application No. W098/07832and in U.S. Pat. No. 6,235,713. These documents also describe theisolation of a biologically active fragment of VEGF-D which consists ofVEGF-D amino acid residues 93 to 201. The VEGF-D gene is broadlyexpressed in the adult human, but is certainly not ubiquitouslyexpressed. VEGF-D is strongly expressed in heart, lung and skeletalmuscle. Intermediate levels of VEGF-D are expressed in spleen, ovary,small intestine and colon, and a lower expression occurs in kidney,pancreas, thymus, prostate and testis. No VEGF-D mRNA was detected inRNA from brain, placenta, liver or peripheral blood leukocytes.

[0014] PlGF was isolated from a term placenta cDNA library. Itsisolation and characteristics are described in detail in Maglione etal., Proc. Natl. Acad. Sci. USA, 88: 9267-9271, (1991). Presently itsbiological function is not well understood.

[0015] VEGF3 was isolated from a cDNA library derived from colon tissue.VEGF3 is stated to have about 36% identity and 66% similarity to VEGF.The method of isolation of the gene encoding VEGF3 is unclear and nocharacterization of the biological activity is disclosed inInternational Patent Application No. PCT/US95/07283 (WO 96/39421).

[0016] Similarity between two proteins is determined by comparing theamino acid sequence and conserved amino acid substitutions of one of theproteins to the sequence of the second protein, whereas identity isdetermined without including the conserved amino acid substitutions.

[0017] As noted above, the PDGF/VEGF family members act primarily bybinding to receptor tyrosine kinases. In general, receptor tyrosinekinases are glycoproteins, which consist of an extracellular domaincapable of binding a specific growth factor(s), a transmembrane domain,which is usually an alpha-helical portion of the protein, ajuxtamembrane domain, which is where the receptor may be regulated by,e.g., protein phosphorylation, a tyrosine kinase domain, which is theenzymatic component of the receptor and a carboxy-terminal tail, whichin many receptors is involved in recognition and binding of thesubstrates for the tyrosine kinase.

[0018] Five endothelial cell-specific receptor tyrosine kinases havebeen identified, belonging to two distinct subclasses: three vascularendothelial cell growth factor receptors, VEGFR-1 (Flt-1), VEGFR-2(KDR/Flk-1), VEGFR-3 (Flt4), and the two receptors of the Tie family,Tie and Tie-2 (Tek). These receptors differ in their specificity andaffinity. All of them have the intrinsic tyrosine kinase activity whichis necessary for signal transduction.

[0019] The only receptor tyrosine kinases known to bind VEGFs areVEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with highaffinity, and VEGFR-1 also binds PlGF. VEGF-B binds to VEGFR-1 with highaffinity, but not to VEGFR-2 or -3 (Olofsson et al, Proc. Natl. Acad.Sci. USA, 95:11709-11714 (1998)). VEGF-C has been shown to be the ligandfor VEGFR-3, and it also activates VEGFR-2 (Joukov et al, The EMBOJournal 15:290-298 (1996)). VEGF-D binds to both VEGFR-2 and VEGFR-3(Achen et al, Proc. Natl. Acad. Sci. USA 95:548-553 (1998)). A ligandfor Tek/Tie-2 has been described in International Patent Application No.PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. Theligand for Tie has not yet been identified.

[0020] A novel 130-135 kDa VEGF isoform specific receptor also has beenpurified and cloned (Soker et al, Cell 92:735-745 (1998)). The VEGFreceptor was found to specifically bind the VEGF₁₆₅ isoform via the exon7 encoded sequence, which shows weak affinity for heparin (Soker et al,Cell 92:735-745 (1998)). Surprisingly, the receptor was shown to beidentical to human neuropilin-1 (NP-1), a receptor involved in earlystage neuromorphogenesis. PlGF-2 also appears to interact with NP-1(Migdal et al, J. Biol. Chem. 273:22272-22278 (1998)).

[0021] VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently byendothelial cells. Generally, both VEGFR-1 and VEGFR-2 are expressed inblood vessel endothelia (Oelrichs et al, Oncogene 8:11-18 (1992);Kaipainen et al, J. Exp. Med. 178:2077-2088 (1993); Dumont et al, Dev.Dyn. 203:80-92 (1995); Fong et al, Dev. Dyn. 207:1-10 (1996)) andVEGFR-3 is mostly expressed in the lymphatic endothelium of adulttissues (Kaipainen et al, Proc. Natl. Acad. Sci. USA 9:3566-3570(1995)). VEGFR-3 is also expressed in the blood vasculature surroundingtumors.

[0022] Although VEGFR-1 is mainly expressed in endothelial cells duringdevelopment, it can also be found in hematopoetic precursor cells duringearly stages of embryogenesis (Fong et al, Nature 376:66-70 (1995)). Inadults, monocytes and macrophages also express this receptor (Barleon etal, Blood 87:3336-3343 (1995)). In embryos, VEGFR-1 is expressed bymost, if not all, vessels (Breier et al, Dev. Dyn. 204:228-239 (1995);Fong et al, Dev. Dyn. 207:1-10 (1996)).

[0023] Since the identification and characterization of VEGF, a numberof important findings have focused attention on the activity ofangiogenic factors and the elucidation of new factors. The earlyfindings showed that angiogenesis is required for normal development andphysiology. Processes such as embryogenesis, wound healing, and corpusluteum formation, all involve angiogenesis and angiogenic factors.During wound healing, for example, VEGF mRNA levels increase suggestinga direct correlation between the expression of VEGF and the healingprocess. Also, a defect in VEGF regulation might be associated withwound healing disorders (Frank, S., et al, J. Biol. Chem.2705:12607-12613 (1995)).

[0024] Another important finding involves the connection betweenangiogenesis and tumor development. Both tumor growth and metastasis areangiogenesis-dependent processes (Folkman, J. and Shing, Y., J. Biol.Chem. 267: 10931-10934 (1992)). For example, when tumor cells areintroduced into an animal, the expression pattern of VEGF mRNA revealsexpression at the highest level in cells at the periphery of necrotic,tumor growth areas. Numerous blood vessels were identified within theseareas. The expression of VEGF in these areas suggests that hypoxemia, astate of deficient oxygenation, triggers expression and release of VEGFin the necrotic tumor. The expression of VEGF-B also has been directlycorrelated with tumor growth (see U.S. Pat. No. 5,840,693). VEGF-Bexpression is especially up regulated in tumor-associated macrophagesand also in ovarian epithelial tumors (Sowter et al, Lab Invest.77:607-14, (1997)). VEGF-B mRNA can be detected in most tumor cell linesinvestigated, including adenocarcinoma, breast carcinoma, lymphoma,squamous cell carcinoma, melanoma, fibrosarcoma and Schwannoma (Salvenet al, Am J Pathol. 153:103-8 (1998)).

[0025] It has been shown that members of the VEGF/PDGF family producevariant transcripts. VEGF has been shown to display differenttranscripts because of alternative splicing. The human VEGF gene hasfive different mRNA species (Neufeld et al, FASEB J. 13:9-22 (1999)),resulting in proteins differing in their molecular mass and biologicalproperties (Carmeliet, P., Nat. Med. 6:389-395 (2000)). The hVEGF-A₁₆₅isoform is the predominant transcript in most human tissues, giving riseto a polypeptide with affinity to the neuropilin-1 receptor, besides thebinding to VEGFR1 and VEGFR2. The hVEGF₁₂₁ and hVEGF₁₈₉ isoforms areexpressed in normal tissues at lower levels. The hVEGF₂₀₆ isoform ismainly expressed in embryonic tissues (Houck et al, Mol Endocrinol.5:1806-14 (1991)), while hVEGF₁₄₅ can only be found in tumor cell lines(Poltorak et al, J Biol Chem. 272:7151-8 (1997)). Moreover, VEGF is alsoregulated in an isoform-specific way under pathological conditions. Inlung and colon carcinomas, hVEGF₁₆₅ and hVEGF₁₂₁ are up-regulated,whereas hVEGF₁₈₉ is not changed, suggesting an isoform-specific role ofVEGF in malignancy (Cheung et al, Hum Pathol. 29:910-4 (1998)). Anisoform specific VEGF targeting experiment with murine VEGF-B has shownthat mVEGF₁₆₄ and mVEGF₁₈₈ are more important for postnatal growth andmaintenance of normal function of cardiovascular system, while mVEGF₁₂₀initiates and promotes vasculogenesis (Carmeliet et al, Nat Med.5:495-502 (1999)).

[0026] The placenta growth factor (PlGF) has three different isoforms,which are expressed in a tissue and development specific way (Maglioneet al, Oncogene 8:925-31 (1993); Cao et al, Biochem Biophys Res Commun.235:493-8 (1997)).

[0027] Two isoforms of VEGF-B, generated by alternative splicing ofmRNA, have been recognized (Grimmond et al, Genome Res. 6:124-131(1996); Olofsson et al, J. Biol. Chem. 271:19310-19317 (1996); Townsonet al, Biochem. Biophys. Res. Commun. 220:922-928 (1996)). They are acell associated form of 167 amino acid residues (VEGF-B₁₆₇) and asecreted form of 186 amino acid residues (VEGF-B₁₈₆). The isoforms havean identical N-terminal domain of 115 amino acid residues, excluding thesignal sequence. The common N-terminal domain is encoded by exons 1-5.Differential use of the remaining exons 6A, 6B and 7 gives rise to thetwo splice isoforms. By the use of an alternative splice-acceptor sitein exon 6, an insertion of 101 bp introduces a frame-shift and a stop ofthe coding region of VEGF-B₁₆₇ cDNA. Thus, the two VEGF-B isoforms havediffering C-terminal domains.

[0028] The different C-terminal domains of the two splice isoforms ofVEGF-B affect their biochemical and cell biological properties. TheC-terminal domain of VEGF-B₁₆₇ is structurally related to thecorresponding region in VEGF, with several conserved cysteine residuesand stretches of basic amino acid residues. Thus, this domain is highlyhydrophilic and basic and, accordingly, VEGF-B₁₆₇ will remaincell-associated on secretion, unless the producing cells are treatedwith heparin or high salt concentrations. The cell-associated moleculesbinding VEGF-B₁₆₇ are likely to be cell surface or pericellular heparinsulfate proteoglycans. It is likely that the cell-association of thisisoform occurs via its unique basic C-terminal region.

[0029] The C-terminal domain of VEGF-B₁₈₆ has no significant similaritywith known amino acid sequences in the databases. The hydrophobiccharacter of the C-terminal domain of VEGF-B₁₈₆ contrasts with theproperties of the hydrophilic and basic C-terminal domain of VEGF-B₁₆₇This is supported by the observation that VEGF-B₁₈₆ does not remaincell-associated on its secretion. Recent evidence indicates that thisisoform is proteolytically processed, which regulates the biologicalproperties of the protein (Olofsson et al, Proc. Natl. Acad. Sci. USA,95:11709-11714 (1998)).

[0030] A further difference is found in the glycosylation of the VEGF-Bisoforms. VEGF-B₁₆₇ is not glycosylated at all, whereas VEGF-B₁₈₆ isO-glycosylated but not N-glycosylated.

[0031] Both isoforms of VEGF-B also form heterodimers with VEGF,consistent with the conservation of the eight cysteine residues involvedin inter- and intramolecular disulfide bonding of PDGF-like proteins.Furthermore, co-expression of VEGF-B and VEGF in many tissues suggeststhat VEGF-B-VEGF heterodimers occur naturally. Heterodimers ofVEGF-B₁₆₇-VEGF remain cell-associated. In contrast, heterodimers ofVEGF-B₁₈₆ and VEGF are freely secreted from cells in a culture medium.VEGF also forms heterodimers with PlGF (DiSalvo, et al, J. Biol. Chem.270:7717-7723 (1995)). The production of heterodimeric complexes betweenthe members of this family of growth factors could provide a basis for adiverse array of angiogenic or regulatory molecules.

[0032] Since the secreted VEGF-B₁₆₇ remains cell-associated, it isintrinsically difficult to obtain significant amounts of solubleVEGF-B₁₆₇. Accordingly, there is a need to develop methods forincreasing the amount of soluble VEGF-B₁₆₇.

SUMMARY OF THE INVENTION

[0033] This invention relates to a N-glycosylated VEGF-B and a methodfor increasing the amount of soluble VEGF-B proteins.

[0034] In a first aspect, the invention provides a purified and isolatednucleic acid molecule having a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:1 (sequence encoding VEGF-B₁₆₇), SEQ IDNO:3 (sequence encoding VEGF-B₁₈₆) and SEQ ID NO:5 (sequence encodingVEGF-B_(Ex1-5)) into which a nucleotide sequence encoding at least oneputative N-glycosylation site has been inserted. The nucleic acidmolecule having said polynucleotide sequence can be naked and/or in avector or liposome. The putative N-glycosylation site is -NXT-, -NXS- or-NXC-, where N represents the amino acid asparagine, X may be any aminoacid, and T, S and C represent the amino acids threonine, serine andcysteine, respectively. The nucleotide sequence which encodes theN-glycosylation site may thus comprise aay-nnn¹-(wgy/wcn)-nnn², with theproviso that -nnn¹- is not tga, tar or cnn, and -nnn²- is preferably notccn, where w represents adenine or thymine/uracil, g represents guanine,y represents cytosine or thymine/uracil, c represents cytosine, nrepresents adenine, cytosine, guanine or thymine/uracil; t representsthymine/uracil, a represents adenine, and r represents guanine oradenine. (Rules for N-glycosylation are described athttp://www.expasy.ch/cgi-bin/nicedoc.pl?PDOC00001). Preferably thenucleotide sequence comprises aay-nnn¹-(agy/wcn)-nnn².

[0035] The invention includes the nucleic acid molecules described aboveas well as fragments of those polynucleotides, and variants of thosepolynucleotides with sufficient similarity to the non-coding strand ofthose polynucleotides to hybridize thereto under stringent conditionsand which can code for VEGF-B or a fragment or analog thereof whichexhibits at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:3 orSEQ ID NO:5 and which binds to VEGFR-1. Thus, such polynucleotidefragments and variants having a nucleotide sequence encoding at leastone putative N-glycosylation site inserted therein are intended asaspects of the invention. Exemplary stringent hybridization conditionsare as follows: hybridization at 42° C. in 5×SSC, 20 mM NaPO₄, pH 6.8,50% formamide; and washing at 42° C. in 0.2×SSC. Those skilled in theart understand that it is desirable to vary these conditions empiricallybased on the length and the GC nucleotide base content of the sequencesto be hybridized, and that well accepted formulas for determining suchvariation exist. See for example Sambrook et al, “Molecular Cloning: ALaboratory Manual”, Second Edition, pages 9.47-9.51, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory (1989).

[0036] Moreover, purified and isolated nucleic acid molecules having apolynucleotide sequence encoding other, non-human, mammalian VEGF-Bforms and having a nucleotide sequence encoding at least one putativeN-glycosylation site inserted therein are aspects of the invention, asare the polypeptides encoded thereby.

[0037] A second aspect of the invention involves the purification andisolation of a protein having an amino acid sequence selected from thegroup consisting of SEQ ID NO:2 (VEGF-B₁₆₇), SEQ ID NO:4 (VEGF-B₁₈₆) andSEQ ID NO:6 (VEGF-B_(Ex1-5)) and having at least one putativeN-glycosylation site inserted therein. The purified and isolated proteinpreferably is produced by the expression of the nucleic acid molecule ofthe invention. As noted above, the at least one putative N-glycosylationsite is -NXT-, -NXS- or NXC, where N represents the amino acidasparagine, X may be any amino acid, and T, S and C represent the aminoacids threonine, serine and cysteine, respectively. Preferably theN-glycosylation site is -NXT- or -NXS-, especially preferably -NXT-. Itis also preferred that X and the amino acid following T or S not beproline.

[0038] As used herein, the term “VEGF-B” collectively refers to theknown VEGF-B167 and VEGF-B186 polypeptide isoforms as well as tofragments or analogs thereof which exhibit at least 90% sequenceidentity to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and which bind toVEGFR-1 and/or have the vasculogenesis stimulating activity of VEGF-B.The active substance preferably will include the amino acid sequencePro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (where Xaa may beany amino acid) which is characteristic of VEGF-B.

[0039] Polypeptides comprising conservative substitutions, insertions,or deletions, but which still retain the biological activity of VEGF-Bare clearly to be understood to be within the scope of the invention.Persons skilled in the art will be well aware of methods which canreadily be used to generate such polypeptides, for example the use ofsite-directed mutagenesis, or specific enzymatic cleavage and ligation.The skilled person will also be aware that peptidomimetic compounds orcompounds in which one or more amino acid residues are replaced by anon-naturally occurring amino acid or an amino acid analog may retainthe required aspects of the biological activity of VEGF-B. Suchcompounds can readily be made and tested by methods known in the art,and are also within the scope of the invention.

[0040] In addition, possible variant forms of the VEGF-B polypeptidewhich may result from alternative splicing, as are known to occur withVEGF and VEGF-B, and naturally-occurring allelic variants of the nucleicacid sequence encoding VEGF-B are encompassed within the scope of theinvention. Allelic variants are well known in the art, and representalternative forms or a nucleic acid sequence which comprisesubstitution, deletion or addition of one or more nucleotides, but whichdo not result in any substantial functional alteration of the encodedpolypeptide.

[0041] Such variant forms of VEGF-B can be prepared by targetingnon-essential regions of the VEGF-B polypeptide for modification. Thesenon-essential regions are expected to fall outside thestrongly-conserved regions of the VEGF/PDGF family of growth factors. Inparticular, the growth factors of the VEGF family, including VEGF-B, aredimeric, and VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A and PDGF-B showcomplete conservation of eight cysteine residues in the N-terminaldomains, i.e. the PDGF/VEGF-homology domains (Olofsson et al., Proc.Natl. Acad. Sci. USA, 1996 93 2576-2581; Joukov et al., EMBO J., 1996 15290-298). These cysteines are thought to be involved in intra- andinter-molecular disulfide bonding. In addition there are furtherstrongly, but not completely, conserved cysteine residues in theC-terminal domains. Loops 1, 2 and 3 of each subunit, which are formedby intra-molecular disulfide bonding, are involved in binding to thereceptors for the PDGF/VEGF family of growth factors (Andersson et al.,Growth Factors, 1995 12 159-164).

[0042] Persons skilled in the art thus are well aware that in most casesthese cysteine residues should be preserved in any proposed variantform, although there may be exceptions since receptor-binding VEGF-Banalogs are known in which one or more of the cysteines is notconserved. Similarly, a skilled worker would be aware that the activesites present in loops 1, 2, and 3 also should be preserved. However,other regions of the molecule can be expected to be of lesser importancefor biological function, and therefore offer suitable targets formodification. Modified polypeptides can readily be tested for theirability to show the biological activity of VEGF-B by routine activityassay procedures such as the endothelial cell proliferation assay.

[0043] Preferably where amino acid substitution is used, thesubstitution is conservative, i.e. an amino acid is replaced by one ofsimilar size and with similar charge properties. As used herein, theterm “conservative substitution” denotes the replacement of an aminoacid residue by another, biologically similar residue, i.e., one thathas similar properties. Examples of conservative substitutions includethe substitution of one hydrophobic residue such as isoleucine, valine,leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan,tyrosine, norleucine or methionine for another, or the substitution ofone polar residue for another, such as the substitution of arginine forlysine, glutamic acid for aspartic acid, or glutamine for asparagine,and the like. Neutral hydrophilic amino acids which can be substitutedfor one another include asparagine, glutamine, serine and threonine. Theterm “conservative substitution” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid. Exemplaryconservative substitutions are set out in the following Table A: TABLE AConservative Substitutions I SIDE CHAIN CHARACTERISTICS AMINO ACIDAliphatic Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R Aromatic H F W Y Other N Q D E

[0044] Alternatively, conservative amino acids can be grouped asdescribed in Lehninger, [Biochemistry, Second Edition; Worth Publishers,Inc. New York, N.Y. (1975), pp.71-77] as set out in the following TableB. TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

[0045] Exemplary conservative substitutions also are set out in thefollowing Table C. TABLE C Conservative Substitutions III OriginalExemplary Residue Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln,Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu(E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu(L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, IlePhe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W)Tyr, Phe Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

[0046] If desired, the VEGF-B proteins of the invention can be modified,for instance, by amidation, carboxylation, or phosphorylation, or by thecreation of acid addition salts, amides, esters, in particularC-terminal esters, and N-acyl derivatives of the peptides of theinvention. The proteins also can be modified to create peptidederivatives by forming covalent or noncovalent complexes with othermoieties. Covalently-bound complexes can be prepared by linking thechemical moieties to functional groups on the side chains of amino acidscomprising the peptides, or at the N- or C-terminus.

[0047] In particular, it is anticipated that the VEGF-B proteins can beconjugated to a reporter group, including, but not limited to aradiolabel, a fluorescent label, an enzyme (e.g., that catalyzes acolorimetric or fluorometric reaction), a substrate, a solid matrix, ora carrier (e.g., biotin or avidin). The polypeptide can be linked to anepitope tag, such as the FLAG® octapeptide (Sigma, St. Louis, Mo.) orhistidine, to assist in affinity purification. Also the polypeptidesaccording to the invention may be labeled with a detectable label. Thepolypeptide may be covalently or non-covalently coupled to a suitablesupermagnetic, paramagnetic, electron dense, ecogenic or radioactiveagent for imaging. For use in diagnostic assays, radioactive ornon-radioactive labels may be used. Examples of radioactive labelsinclude a radioactive atom or group, such as ¹²⁵I or ³²p. Examples ofnon-radioactive labels include enzymatic labels, such as horseradishperoxidase or fluorimetric labels, such as fluorescein-5-isothiocyanate(FITC). Labeling may be direct or indirect, covalent or non-covalent.

[0048] The modified polypeptides can readily be tested for their abilityto show the biological activity of VEGF-B by routine activity assayprocedures such as the fibroblast proliferation assay.

[0049] It will be clearly understood that nucleic acids and polypeptidesof the invention may be prepared by synthetic means or by recombinantmeans, or may be purified from natural sources.

[0050] As used herein, the term “comprising” means “included but notlimited to”. The corresponding meaning applies to the word “comprises”.

[0051] A third aspect of the invention provides vectors comprising thenucleic acid molecule of the first aspect of the invention, and hostcells transformed or transfected with nucleic acids molecules or vectorsof the invention. These may be eukaryotic or prokaryotic in origin.These cells are particularly suitable for expression of the polypeptideof the invention, and include insect cells such as Sf9 or HF cells,obtainable from the American Type Culture Collection, infected with arecombinant baculovirus, and the human embryo kidney cell line 293-EBNAtransfected by a suitable expression plasmid. Preferred vectors of theinvention are expression vectors in which a nucleic acid according tothe invention is operatively connected to one or more appropriatepromoters and/or other control sequences, such that appropriate hostcells transformed or transfected with the vectors are capable ofexpressing the polypeptide of the invention. Other preferred vectors arethose suitable for transfection of mammalian cells, or for gene therapy,such as adenoviral-, vaccinia- or retroviral-based vectors or liposomes.A variety of such vectors are known in the art.

[0052] The invention also provides a method of making a vector capableof expressing a polypeptide encoded by a nucleic acid according to theinvention, comprising the steps of operatively connecting the nucleicacid molecule of the first aspect to one or more appropriate promotersand/or other control sequences, as described above.

[0053] The invention further provides a method of making a polypeptideaccording to the invention, comprising the steps of expressing a nucleicacid or vector of the invention in a host cell, and isolating thepolypeptide from the host cell or from the host cell's growth medium.

[0054] The polypeptide according to the invention may be employed incombination with a suitable pharmaceutical carrier. The resultingcompositions comprise an effective amount of glycosylated VEGF-B or apharmaceutically acceptable non-toxic salt thereof, and apharmaceutically acceptable solid or liquid carrier or adjuvant.Examples of such a carrier or adjuvant include, but are not limited to,saline, buffered saline, Ringer's solution, mineral oil, talc, cornstarch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, sodium chloride, alginic acid, dextrose,water, glycerol, ethanol, thickeners, stabilizers, suspending agents andcombinations thereof. Such compositions may be in the form of solutions,suspensions, tablets, capsules, creams, salves, elixirs, syrups, wafers,ointments or other conventional forms. The formulation to suit the modeof administration. Compositions can comprise a glycosylated VEGF-B andoptionally further comprise one or more of PDGF-A, PDGF-B, VEGF,non-glycosylated VEGF-B, VEGF-C, VEGF-D, PlGF and/or heparin.Compositions comprising the glycosylated VEGF-B will contain from about0.1% to 90% by weight of the active compound(s), and most generally fromabout 10% to 30%.

[0055] For intramuscular preparations, a sterile formulation, preferablya suitable soluble salt form of the glycosylated VEGF-B, such ashydrochloride salt, can be dissolved and administered in apharmaceutical diluent such as pyrogen-free water (distilled),physiological saline or 5% glucose solution. A suitable insoluble formof the compound may be prepared and administered as a suspension in anaqueous base or a pharmaceutically acceptable oil base, e.g. an ester ofa long chain fatty acid such as ethyl oleate.

[0056] In a further aspect, the invention provides a method for making asoluble VEGF-B₁₆₇ from a host cell and a method for increasing an amountof a soluble VEGF-B₁₆₇ ₁ VEGF-B₁₈₆ or VEGF-B_(Ex1-5) protein from a hostcell. These methods comprise inserting at least one putativeN-glycosylation site into a nucleotide sequence which codes forVEGF-B₁₆₇, VEGF-B₁₈₆ or VEGF-BE_(EX1-5) protein; transforming ortransfecting said nucleotide sequence with the inserted N-glycosylationsite into a host cell; culturing the transfected host cell in a growthmedium such that said nucleotide sequence with inserted N-glycosylationsite is expressed; and isolating the expressed polypeptide from thegrowth medium in which said host cell was cultured. These methods canfurther comprise exposing the cultured transfected host cell to heparinafter said polypeptide is expressed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The invention will be described in further detail hereinafterwith reference to the accompanying drawings in which:

[0058]FIG. 1 is an alignment of the amino acid sequences of the VEGFhomology domain (VHD) of VEGF-A and PlGF with VEGF-B;

[0059]FIG. 2 is a diagram of plasmid pSecTagA-hVEGF-B₁₈₆-H₆-NXTcontaining a nucleotide sequence encoding VEGF-B₁₈₆ having anN-glycosylation site incorporated therein;

[0060]FIG. 3 is a diagram of plasmid pSecTagA-hVEGF-B₁₆₇-H₆-NXTcontaining a nucleotide sequence encoding VEGF-B₁₆₇ having anN-glycosylation site incorporated therein;

[0061]FIG. 4 is a diagram of plasmid pSecTagA-hVEGF-B-Exon1-5-H₆-NXTcontaining a nucleotide sequence encoding exons 1-5 of VEGF-B having anN-glycosylation site incorporated therein;

[0062]FIG. 5 shows the expression of hVEGF-B₁₆₇ with and without thepotential glycosylation site (NXT);

[0063]FIG. 6 shows the expression of hVEGF-B₁₆₇ and hVEGF-B₁₈₆ with andwithout the potential glycosylation site (NXT);

[0064]FIG. 7 shows the expression and receptor binding of hVEGF-B₁₆₇ andhVEGF-B₁₈₆ with and without the potential glycosylation site (NXT); and

[0065]FIG. 8 shows the expression and receptor binding of polypeptideencoded by exons 1-5 of hVEGF-B with and without the potentialglycosylation site (NXT).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0066] Introduction of the Glycosylation Site

[0067] As mentioned before, VEGF-B is a PDGF/VEGF family member that iscompletely devoid of any N-glycosylation. To analyze the effects ofN-glycosylation on VEGF-B, a N-glycosylation site was introduced intoVEGF-B. To determine the most appropriate site to introduce a mutationthat would lead to N-glycosylation of VEGF-B, the amino acid sequencesof the first 99 amino acids of VEGF-A, PlGF and VEGF-B, respectively,were aligned (see FIG. 1). The N-glycosylation sites of VEGF-A and PlGFat amino acids 65-67 are italicized in FIG. 1. Nucleotides encoding aputative N-glycosylation site (NXT) were inserted at the positioncorresponding to nucleotides 286-294 of hVEGF-B (SEQ ID NO:1). Thereplaced nucleotides normally found at positions 286-294 encode theamino acid residues QVR and these amino acid residues are bolded in FIG.1.

EXAMPLE 2

[0068] Preparation of Recombinant Vectors

[0069] Six mammalian expression vectors for both naturally occurringisoforms of VEGF-B (i.e., VEGF-B₁₆₇ and VEGF-B₁₈₆) and for an artificialsplice variant (comprising exons 1 to 5 only) were constructed with andwithout the putative N-glycosylation site.

[0070] Using PCR, nucleotides coding for a histidine tag were added tothe C-terminal end of a nucleotide sequence coding for hVEGF-B₁₈₆. Anucleotide sequence coding for hVEGF-B₁₈₆-H₆ was then inserted intopSecTagA (Invitrogen, Carlsbad, Calif.) using standard cloningprocedures to construct pSecTagA-hVEGF-B₁₈₆-H₆. The full sequence ofpSecTagA-hVEGF-B₁₈₆-H₆ is given in SEQ ID NO:7.

[0071] To construct pSecTagA-hVEGF-B₁₈₆-H₆-NXT, a PCR product ofcovering nucleotides 1-325 from Genebank Acc. No. U48801 was producedwhich introduced a N-glycosylation site at nucleotide positions 289-297using the 3′ primer:5′-TCGGTACCGGATCATGAGGATCTGCATGGTGACGTTGTGCTGCCCAGTGGCCA-3′ (SEQ IDNO:8). This PCR product was then cloned into a plasmid with full-lengthhVEGF-B₁₈₆ where it used to replace the corresponding sequence toproduce hVEGF-B₁₈₆-NXT. A histidine tag was then added by cloningtogether the N-terminal portion of hVEGF-B₁₈₆-NXT with the C-terminalportion of hVEGF-B₁₈₆-H₆ using standard cloning procedures to producehVEGF-B₁₈₆-H₆-NXT. The nucleotide sequence coding for hVEGF-B₁₈₆-H₆-NXTwas then inserted into pSecTagA (Invitrogen) using standard cloningprocedures to construct pSecTagA-hVEGF-B₁₈₆-H₆-NXT. The full sequence ofpSecTagA-hVEGF-B₁₈₆-H₆-NXT is given in SEQ ID NO:9, and the plasmid isillustrated in FIG. 2.

[0072] To construct pSecTagA-hVEGF-B₁₆₇-H₆, a 349 bp PCR product wasproduced covering nucleotides 250-567 from Genebank Acc. No. U48801,nucleotides coding for the histidine tag, a stop codon, the NotIrestriction site and terminal clamp nucleotides using the 5′ primer:5′-CCTGACGATGGCCTGGAGTGT-3′ (SEQ ID NO:10) and the 3′ primer:5′-GAGCGGCCGCTCAATGATGATGATGATGATGCCTTCGCAGCTTCCGGCAC-3′ (SEQ ID NO:11)and hVEGF-B₁₆₇ as the template. The 349 bp PCR product was cut with KpnIand NotI and the KpnI-NotI fragment was inserted intopSecTagA-hVEGF-B₁₈₆-H₆ to replace the KpnI-NotI fragment removed fromthis vector using standard cloning procedures. The full sequence ofpSecTagA-hVEGF-B₁₆₇-H₆ is given in SEQ ID NO:12.

[0073] Similarly, pSecTagA-hVEGF-B₁₆₇-H₆-NXT was constructed as aboveexcept the KpnI-NotI fragment was inserted intopSecTagA-hVEGF-B₁₈₆-H₆-NXT to replace the KpnI-NotI fragment removedfrom this vector. The full sequence of pSecTagA-hVEGF-B₁₆₇-H₆-NXT isgiven in SEQ ID NO:13, and the plasmid is illustrated in FIG. 3.

[0074] To construct pSecTagA-hVEGF-B_(Ex1-5)-H₆, a 443 bp PCR productwas obtained covering nucleotides 1-411 from Genebank Acc. No. U48801,nucleotides coding for the histidine tag, a stop codon, the NotIrestriction site and terminal clamp nucleotides using the 5′ primer:5′-CACCATGAGCCCTCTGCTCC-3′ (SEQ ID NO:14) and 3′ primer:5-GAGCGGCCGCTCAGTGGTGATGATGATGGTCTGGCTTCACAGCACTG-3′ (SEQ ID NO:15) andhVEGF-B₁₆₇ as the template. The PCR product was cut with KpnI and NotIand the resulting 320 bp fragment was inserted intopSecTagA-hVEGF-B₁₈₆-H₆-NXT to replace the KpnI-NotI removed from thisvector using standard cloning procedures. The full sequence ofpSecTagA-hVEGF-B_(Ex1-5)-H₆ is given in SEQ ID NO:16.

[0075] To construct pSecTagA-hVEGF-B_(Ex1-5)-H₆-NXT, the same proceduresas above were used except the KpnI-NotI fragment was inserted intopSecTagA-hVEGF-B₁₈₆-H₆-NXT to replace the KpnI-NotI fragment removedfrom this vector. The full sequence of pSecTagA-hVEGF-B_(Ex1-5) -H₆-NXTis given in SEQ ID NO:17, and the plasmid is illustrated in FIG. 4.

[0076] The following Table D lists the expression vectors for thenaturally occurring 186 and 167 amino acid isoforms of VEGF-B and forthe artificial splice variant (comprising exon 1 to 5 only), constructedwith and without the potential glycosylation site (NXT). TABLE DConstruct Name Protein pSecTagA-hVEGF-B₁₈₆-H₆ histidine-tagged VEGF-B₁₈₆psecTagA-hVEGF-B₁₈₆-H₆-NXT histidine-tagged and N-glycosylated VEGF-B₁₈₆pSecTagA-hVEGF-B₁₆₇-H₆ histidine-tagged VEGF-B₁₆₇pSecTagA-hVEGF-B₁₆₇-H₆-NXT histidine-tagged and N-glycosylated VEGF-B₁₆₇pSecTagA-hVEGF-B-Exon1-5-H₆ histidine-tagged VEGF-B Exons 1 to 5 onlypSecTagA-hVEGF-B-Exon1-5-H₆-NXT histidine-tagged and N-glycosylatedVEGF-B Exons 1 to 5 only

EXAMPLE 3

[0077] Transfection and Expression of Recombinant Proteins

[0078] The six mammalian expression vectors of human VEGF-B describedabove along with expression vectors containing histidine-tagged VEGF(positive control), a histidine-tagged VHD of VEGF-C (negative control)and histidine-tagged hybrid 84-11 (positive control), respectively, weretransfected into 293T cells using CaPO₄-mediated transfection accordingto procedures described in Sambrook, J. et al., Molecular Cloning, ALaboratory Manual, (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.),16.33-16.36 (1989). 48 hours after transfection, the cells weremetabolically labeled with S³⁵ methionine and S³⁵ cysteine (Promix,Amersham) for 12 to 24 hours. The conditioned supernatant was subjectedto immunoprecipitation with an antiserum specific to human VEGF-B (874)and a monoclonal antibody specific to pentahistidine (H₅ mAb, Qiagen).

[0079] As seen in FIGS. 5 through 8, the three constructs produced withthe inserted putative N-glycosylation site are glycosylated.

[0080] As can be seen from FIGS. 5-7, comparison of supernatants andlysates and using heparin to release cell bound proteins shows thatVEGF-B₁₆₇ is almost completely retained at the cell surface or withinthe cell. About a 50 fold increase of VEGF-B₁₆₇ can be detected in thesupernatant upon glycosylation (FIG. 5). As shown in FIGS. 6 and 7,VEGF-B₁₆₇ is released into the supernatant by treatment with 100 μg/mlheparin two hours prior to harvest. It was also found that approximatelytwo times more glycosylated VEGF-B₁₆₇ can be detected in the supernatantof non-heparin treated 293T cells as compared to non-glycosylatedVEGF-B₁₆₇ treated with 200 μg/ml heparin for two hours prior toharvesting. In addition, there is about a three fold increase in theamount of the glycosylated VEGF-B₁₆₇ detected in the supernatant bytreatment with 200 μg/ml heparin two hours prior to harvest as comparedto glycosylated VEGF-B₁₆₇ without heparin treatment, and approximately asix fold increase in the amount of the glycosylated VEGF-B₁₆₇ detectedin the supernatant by treatment with 200 μg/ml heparin two hours priorto harvest as compared to the amount of non-glycosylated VEGF-B₁₆₇detected in the supernatant with the same heparin treatment.

[0081]FIGS. 6 and 7 show that VEGF-B₁₈₆ is also partially retained atthe cell surface and within the cell. In contrast to VEGF-B₁₆₇, almostall of the VEGF-B₁₈₆ is released into the supernatant upon glycosylationand heparin treatment (FIGS. 6 and 7). There seems to be no significantdifference in the amount of VEGF-B₁₈₆ found in the supernatant betweenheparin-treated and untreated 293T cells. Thus the difference ofVEGF-B₁₈₆ and N-glycosylated VEGF-B₁₈₆ protein levels in the supernatant(approximately three times more glycosylated VEGF-B₁₈₆) seems to bemainly due to enhanced secretion and/or production and not due to therelease of cell surface bound protein.

[0082]FIG. 8 shows that VEGF-B_(Exon1-5) is only efficiently releasedinto the medium if it is N-glycosylated (over a 50 fold increase insoluble protein). This is unexpected since the signals retaining VEGF-Bat the cell surface are thought to reside in the exon 6 and 7 encodeddomains (FIG. 8). Treatment with heparin was not determined for thissame reason.

EXAMPLE 4

[0083] VEGF Receptor 1 Binding of Recombinant Proteins

[0084] The ability of the recombinant VEGF-B to bind VEGF receptor 1(VEGFR-1) was analyzed using soluble fusion proteins consisting of theextracellular domain of VEGFR-1 and the Fc portion of human IgG1(VEGFR-1-Fc). Biosynthetically labeled conditioned medium derived from293T cells transfected as above in Example 3 were immunoprecipitatedwith protein A sepharose (PAS) bound to the VEGFR-1-Ig. Beads werewashed three times with PBS, the bound protein eluted and resolved byreducing SDS-PAGE (15%). The dried gels were exposed to phosphoimagerplates for 12-24 hours. Additionally, the cell lysates wereimmunoprecipitated with H₅ mAb.

[0085] When significant amounts of VEGF-B were present in thesupernatant, binding to VEGFR-1 could be observed. This was seen withVEGF-B₁₈₆-H₆ after treatment with 100 μg/ml heparin two hours prior toharvest, VEGF-B₁₈₆-NXT-H₆ and VEGF-B Exon 1-5-NXT-H₆ (FIGS. 7 and 8).

EXAMPLE 5

[0086] Stimulation of BaF3 VEGFR-01EC/EpoR Cell Survival

[0087] The effects of introducing the N-glycosylation site into VEGF-Bcan be assayed by measuring the ability of conditioned media from cellstransfected with VEGF-B167 and VEGF-B167-NXT and/or VEGF-B186 andVEGF-B186-NXT to stimulate the survival of BaF3 VEGFR-01EC/EpoR cells.For the assay, BaF3 cells are used that are stably transfectd with achimeric receptor consisting of the extracellular domain of VEGFreceptor 1 and the intracellular domain of the erythropoietin receptor.For survival, these cells need either interlukin-3 or any growth factorcapable of binding VEGFR-1, e.g., VEGF-A, VEGF-B or PlGF. Cells areplated to 96-well plates at a density of 20,000/well and grown in thepresence of different amounts of medium conditioned by 293T cells thathave been transfected with VEGF-B167 and VEGF-B167-NXT, VEGF-B186 andVEGF-B186-NXT, or both. Conditioned medium from 293T cells transfectedwith a mock (i.e., empty) vector may be used as a control. Prior to theassay, the conditioned medium should be cleared from potentiallyinterfering proteins by immunoprecipitation using appropriateantibodies. For example, VEGF-A may be cleared from the conditionedmedium prior to the assay using a mixture of monoclonal and polyclonalanti-hVEGF antibodies, commercially available from R&D Systems,Minneapolis, Minn. It is not necessary to preclear medium of PlGF as theamounts expressed by COS cells (if any) are negligible and its effectsare not visible in the baseline noise. After 48 hours, an MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide thiazoleblue) colorimetric assay may be performed and data collected at 540 nmusing a microtitreplate reader.

[0088] To create the BglII site in the coding sequence of human VEGFR-1just before the transmembrane domain, basepairs 1998-2268 of VEGFR-1were PCR amplified with primers 5′-CCTCAGTGATCACACAGTGG-3′, containingthe endogenous BclI site, and 5′-CAGAGATCTATTAGACTTGTCC-3′, containing aBglII site, and the PCR fragment was cloned into the BclI-BglII sites ofVEGFR-1 in pBlueScript SKII+ (Stratagene) vector. The transmembrane andintracellular domains of the human erythropoietin receptor were excisedfrom EpoR×B+B/pcDNAI and subcloned into the resulting plasmid as aBglII/NotI fragment. The EpoR×B+B is a clone of EpoR which has aninternal BglII site added at the putative transmembrane(TM)/extracellular (EC) domain junction for the in-frame ligation of RTKextracellular domains. The vector backbone is pCDNA1-amp (˜5.4 kb, theoriginal 1.75 kb EpoR clone was subcloned into pCDNA1-amp using KpnI,the sequence was reported by the Lodish Laboratory, MIT). An ˜1 kbfragment can be excised from this clone using BglII (5′)- NotI (3′)digest which contains the TM and cytoplasmic domain of EpoR.

[0089] The VEGFR-1/EpoR construct was finally subcloned into the pEF-BOSvector (Mizushima et al., Nucleic Acids Research, 18(17):5322 Sep. 11,1990) as a KpnI/NotI fragment. The resulting plasmid was electroporatedinto BaF3 cells and stable cell pools were generated by selection with250 micrograms/mL zoecin.

[0090] The foregoing description and examples have been set forth merelyto illustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include everything within thescope of the appended claims and equivalents thereof.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 17 <210> SEQ ID NO 1<211> LENGTH: 567 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(567) <221> NAME/KEY:mat_peptide <222> LOCATION: (64)..(564) <400> SEQUENCE: 1 atg agc cctctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctg cag ctg 48 Met Ser Pro LeuLeu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu -20 -15 -10 gcc ccc gcccag gcc cct gtc tcc cag cct gat gcc cct ggc cac cag 96 Ala Pro Ala GlnAla Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln -5 -1 1 5 10 agg aaa gtggtg tca tgg ata gat gtg tat act cgc gct acc tgc cag 144 Arg Lys Val ValSer Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 15 20 25 ccc cgg gag gtggtg gtg ccc ttg act gtg gag ctc atg ggc acc gtg 192 Pro Arg Glu Val ValVal Pro Leu Thr Val Glu Leu Met Gly Thr Val 30 35 40 gcc aaa cag ctg gtgccc agc tgc gtg act gtg cag cgc tgt ggt ggc 240 Ala Lys Gln Leu Val ProSer Cys Val Thr Val Gln Arg Cys Gly Gly 45 50 55 tgc tgc cct gac gat ggcctg gag tgt gtg ccc act ggg cag cac caa 288 Cys Cys Pro Asp Asp Gly LeuGlu Cys Val Pro Thr Gly Gln His Gln 60 65 70 75 gtc cgg atg cag atc ctcatg atc cgg tac ccg agc agt cag ctg ggg 336 Val Arg Met Gln Ile Leu MetIle Arg Tyr Pro Ser Ser Gln Leu Gly 80 85 90 gag atg tcc ctg gaa gaa cacagc cag tgt gaa tgc aga cct aaa aaa 384 Glu Met Ser Leu Glu Glu His SerGln Cys Glu Cys Arg Pro Lys Lys 95 100 105 aag gac agt gct gtg aag ccagac agc ccc agg ccc ctc tgc cca cgc 432 Lys Asp Ser Ala Val Lys Pro AspSer Pro Arg Pro Leu Cys Pro Arg 110 115 120 tgc acc cag cac cac cag cgccct gac ccc cgg acc tgc cgc tgc cgc 480 Cys Thr Gln His His Gln Arg ProAsp Pro Arg Thr Cys Arg Cys Arg 125 130 135 tgc cga cgc cgc agc ttc ctccgt tgc caa ggg cgg ggc tta gag ctc 528 Cys Arg Arg Arg Ser Phe Leu ArgCys Gln Gly Arg Gly Leu Glu Leu 140 145 150 155 aac cca gac acc tgc aggtgc cgg aag ctg cga agg tga 567 Asn Pro Asp Thr Cys Arg Cys Arg Lys LeuArg Arg 160 165 <210> SEQ ID NO 2 <211> LENGTH: 188 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Ser Pro Leu Leu ArgArg Leu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln AlaPro Val Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val SerTrp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val ValVal Pro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu ValPro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro AspAsp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met GlnIle Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met SerLeu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys AspSer Ala Val Lys Pro Asp Ser Pro Arg Pro Leu Cys Pro Arg 130 135 140 CysThr Gln His His Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg 145 150 155160 Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu 165170 175 Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg 180 185 <210>SEQ ID NO 3 <211> LENGTH: 624 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(621)<221> NAME/KEY: mat_peptide <222> LOCATION: (64)..(621) <400> SEQUENCE:3 atg agc cct ctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctg cag ctg 48 MetSer Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu -20 -15 -10gcc ccc gcc cag gcc cct gtc tcc cag cct gat gcc cct ggc cac cag 96 AlaPro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln -5 -1 1 5 10agg aaa gtg gtg tca tgg ata gat gtg tat act cgc gct acc tgc cag 144 ArgLys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 15 20 25 ccccgg gag gtg gtg gtg ccc ttg act gtg gag ctc atg ggc acc gtg 192 Pro ArgGlu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val 30 35 40 gcc aaacag ctg gtg ccc agc tgc gtg act gtg cag cgc tgt ggt ggc 240 Ala Lys GlnLeu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 45 50 55 tgc tgc cctgac gat ggc ctg gag tgt gtg ccc act ggg cag cac caa 288 Cys Cys Pro AspAsp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 60 65 70 75 gtc cgg atgcag atc ctc atg atc cgg tac ccg agc agt cag ctg ggg 336 Val Arg Met GlnIle Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 80 85 90 gag atg tcc ctggaa gaa cac agc cag tgt gaa tgc aga cct aaa aaa 384 Glu Met Ser Leu GluGlu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 95 100 105 aag gac agt gctgtg aag cca gac agg gct gcc act ccc cac cac cgt 432 Lys Asp Ser Ala ValLys Pro Asp Arg Ala Ala Thr Pro His His Arg 110 115 120 ccc cag ccc cgttct gtt ccg ggc tgg gac tct gcc ccc gga gca ccc 480 Pro Gln Pro Arg SerVal Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro 125 130 135 tcc cca gct gacatc acc cat ccc act cca gcc cca ggc ccc tct gcc 528 Ser Pro Ala Asp IleThr His Pro Thr Pro Ala Pro Gly Pro Ser Ala 140 145 150 155 cac gct gcaccc agc acc acc agc gcc ctg acc ccc gga cct gcc gcc 576 His Ala Ala ProSer Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala Ala 160 165 170 gcc gct gccgac gcc gca gct tcc tcc gtt gcc aag ggc ggg gct tag 624 Ala Ala Ala AspAla Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 175 180 185 <210> SEQ ID NO4 <211> LENGTH: 207 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 4 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu GlnLeu -20 -15 -10 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro GlyHis Gln -5 -1 1 5 10 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg AlaThr Cys Gln 15 20 25 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu MetGly Thr Val 30 35 40 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln ArgCys Gly Gly 45 50 55 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr GlyGln His Gln 60 65 70 75 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro SerSer Gln Leu Gly 80 85 90 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu CysArg Pro Lys Lys 95 100 105 Lys Asp Ser Ala Val Lys Pro Asp Arg Ala AlaThr Pro His His Arg 110 115 120 Pro Gln Pro Arg Ser Val Pro Gly Trp AspSer Ala Pro Gly Ala Pro 125 130 135 Ser Pro Ala Asp Ile Thr His Pro ThrPro Ala Pro Gly Pro Ser Ala 140 145 150 155 His Ala Ala Pro Ser Thr ThrSer Ala Leu Thr Pro Gly Pro Ala Ala 160 165 170 Ala Ala Ala Asp Ala AlaAla Ser Ser Val Ala Lys Gly Gly Ala 175 180 185 <210> SEQ ID NO 5 <211>LENGTH: 408 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(408) <221> NAME/KEY:mat_peptide <222> LOCATION: (64)..(408) <400> SEQUENCE: 5 atg agc cctctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctg cag ctg 48 Met Ser Pro LeuLeu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu -20 -15 -10 gcc ccc gcccag gcc cct gtc tcc cag cct gat gcc cct ggc cac cag 96 Ala Pro Ala GlnAla Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln -5 -1 1 5 10 agg aaa gtggtg tca tgg ata gat gtg tat act cgc gct acc tgc cag 144 Arg Lys Val ValSer Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 15 20 25 ccc cgg gag gtggtg gtg ccc ttg act gtg gag ctc atg ggc acc gtg 192 Pro Arg Glu Val ValVal Pro Leu Thr Val Glu Leu Met Gly Thr Val 30 35 40 gcc aaa cag ctg gtgccc agc tgc gtg act gtg cag cgc tgt ggt ggc 240 Ala Lys Gln Leu Val ProSer Cys Val Thr Val Gln Arg Cys Gly Gly 45 50 55 tgc tgc cct gac gat ggcctg gag tgt gtg ccc act ggg cag cac caa 288 Cys Cys Pro Asp Asp Gly LeuGlu Cys Val Pro Thr Gly Gln His Gln 60 65 70 75 gtc cgg atg cag atc ctcatg atc cgg tac ccg agc agt cag ctg ggg 336 Val Arg Met Gln Ile Leu MetIle Arg Tyr Pro Ser Ser Gln Leu Gly 80 85 90 gag atg tcc ctg gaa gaa cacagc cag tgt gaa tgc aga cct aaa aaa 384 Glu Met Ser Leu Glu Glu His SerGln Cys Glu Cys Arg Pro Lys Lys 95 100 105 aag gac agt gct gtg aag ccagac 408 Lys Asp Ser Ala Val Lys Pro Asp 110 115 <210> SEQ ID NO 6 <211>LENGTH: 136 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:6 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu -20-15 -10 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln-5 -1 1 5 10 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr CysGln 15 20 25 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly ThrVal 30 35 40 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys GlyGly 45 50 55 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln HisGln 60 65 70 75 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser GlnLeu Gly 80 85 90 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg ProLys Lys 95 100 105 Lys Asp Ser Ala Val Lys Pro Asp 110 115 <210> SEQ IDNO 7 <211> LENGTH: 5614 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: pSecTagA-VEGF-B167-H6 <400> SEQUENCE: 7 gacggatcgggagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagttaagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaatttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttaggcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgactagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccgcgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccattgacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtcaatgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgccaagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagtacatgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattaccatggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacggggatttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacgggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgtacggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactggcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 900 ccagtgtggtggaattcggc ttcaccatga gccctctgct ccgccgcctg ctgctcgccg 960 cactcctgcagctggccccc gcccaggccc ctgtctccca gcctgatgcc cctggccacc 1020 agaggaaagtggtgtcatgg atagatgtgt atactcgcgc tacctgccag ccccgggagg 1080 tggtggtgcccttgactgtg gagctcatgg gcaccgtggc caaacagctg gtgcccagct 1140 gcgtgactgtgcagcgctgt ggtggctgct gccctgacga tggcctggag tgtgtgccca 1200 ctgggcagcaccaagtccgg atgcagatcc tcatgatccg gtacccgagc agtcagctgg 1260 gggagatgtccctggaagaa cacagccagt gtgaatgcag acctaaaaaa aaggacagtg 1320 ctgtgaagccagacagcccc aggcccctct gcccacgctg cacccagcac caccagcgcc 1380 ctgacccccggacctgccgc tgccgctgcc gacgccgcag cttcctccgt tgccaagggc 1440 ggggcttagagctcaaccca gacacctgca ggtgccggaa gctgcgaagg catcatcatc 1500 atcatcattgagcggccgct cgagtctaga gggcccgaac aaaaactcat ctcagaagag 1560 gatctgaatagcgccgtcga ccatcatcat catcatcatt gagtttaaac ccgctgatca 1620 gcctcgactgtgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 1680 ttgaccctggaaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 1740 cattgtctgagtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 1800 gaggattgggaagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 1860 gcggaaagaaccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 1920 agcgcggcgggtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 1980 cccgctcctttcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 2040 gctctaaatcggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc 2100 aaaaaacttgattagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 2160 cgccctttgacgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 2220 acactcaaccctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc 2280 tattggttaaaaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 2340 tgtgtcagttagggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 2400 tgcatctcaattagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 2460 gtatgcaaagcatgcatctc aattagtcag caaccatagt cccgccccta actccgccca 2520 tcccgcccctaactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 2580 ttatttatgcagaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 2640 gcttttttggaggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 2700 gatctgatcagcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata 2760 cgacaaggtgaggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc 2820 gcgcgacgtcgccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt 2880 cgtggaggacgacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt 2940 ccaggaccaggtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga 3000 gctgtacgccgagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc 3060 catgaccgagatcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg 3120 caactgcgtgcacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc 3180 caccgccgccttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 3240 gatcctccagcgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc 3300 agcttataatggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 3360 ttcactgcattctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat 3420 accgtcgacctctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 3480 ttgttatccgctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 3540 gggtgcctaatgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 3600 gtcgggaaacctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 3660 tttgcgtattgggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3720 gctgcggcgagcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 3780 ggataacgcaggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 3840 ggccgcgttgctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 3900 acgctcaagtcagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 3960 tggaagctccctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4020 ctttctcccttcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4080 ggtgtaggtcgttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4140 ctgcgccttatccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4200 actggcagcagccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4260 gttcttgaagtggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4320 tctgctgaagccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4380 caccgctggtagcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4440 atctcaagaagatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4500 acgttaagggattttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 4560 ttaaaaatgaagttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 4620 ccaatgcttaatcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 4680 tgcctgactccccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 4740 tgctgcaatgataccgcgag acccacgctc accggctcca gatttatcag caataaacca 4800 gccagccggaagggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 4860 tattaattgttgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 4920 tgttgccattgctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 4980 ctccggttcccaacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5040 tagctccttcggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5100 ggttatggcagcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5160 gactggtgagtactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5220 ttgcccggcgtcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 5280 cattggaaaacgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5340 ttcgatgtaacccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 5400 ttctgggtgagcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5460 gaaatgttgaatactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 5520 ttgtctcatgagcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 5580 gcgcacatttccccgaaaag tgccacctga cgtc 5614 <210> SEQ ID NO 8 <211> LENGTH: 5614<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:pSecTagA-VEGF-B167-H6-NXT <400> SEQUENCE: 8 gacggatcgg gagatctcccgatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtatctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctacaacaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcgctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggactatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgaccttatgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa gctggctagt 900 ccagtgtggt ggaattcggcttcaccatga gccctctgct ccgccgcctg ctgctcgccg 960 cactcctgca gctggcccccgcccaggccc ctgtctccca gcctgatgcc cctggccacc 1020 agaggaaagt ggtgtcatggatagatgtgt atactcgcgc tacctgccag ccccgggagg 1080 tggtggtgcc cttgactgtggagctcatgg gcaccgtggc caaacagctg gtgcccagct 1140 gcgtgactgt gcagcgctgtggtggctgct gccctgacga tggcctggag tgtgtgccca 1200 ctgggcagca caacgtcaccatgcagatcc tcatgatccg gtacccgagc agtcagctgg 1260 gggagatgtc cctggaagaacacagccagt gtgaatgcag acctaaaaaa aaggacagtg 1320 ctgtgaagcc agacagccccaggcccctct gcccacgctg cacccagcac caccagcgcc 1380 ctgacccccg gacctgccgctgccgctgcc gacgccgcag cttcctccgt tgccaagggc 1440 ggggcttaga gctcaacccagacacctgca ggtgccggaa gctgcgaagg catcatcatc 1500 atcatcattg agcggccgctcgagtctaga gggcccgaac aaaaactcat ctcagaagag 1560 gatctgaata gcgccgtcgaccatcatcat catcatcatt gagtttaaac ccgctgatca 1620 gcctcgactg tgccttctagttgccagcca tctgttgttt gcccctcccc cgtgccttcc 1680 ttgaccctgg aaggtgccactcccactgtc ctttcctaat aaaatgagga aattgcatcg 1740 cattgtctga gtaggtgtcattctattctg gggggtgggg tggggcagga cagcaagggg 1800 gaggattggg aagacaatagcaggcatgct ggggatgcgg tgggctctat ggcttctgag 1860 gcggaaagaa ccagctggggctctaggggg tatccccacg cgccctgtag cggcgcatta 1920 agcgcggcgg gtgtggtggttacgcgcagc gtgaccgcta cacttgccag cgccctagcg 1980 cccgctcctt tcgctttcttcccttccttt ctcgccacgt tcgccggctt tccccgtcaa 2040 gctctaaatc ggggcatccctttagggttc cgatttagtg ctttacggca cctcgacccc 2100 aaaaaacttg attagggtgatggttcacgt agtgggccat cgccctgata gacggttttt 2160 cgccctttga cgttggagtccacgttcttt aatagtggac tcttgttcca aactggaaca 2220 acactcaacc ctatctcggtctattctttt gatttataag ggattttggg gatttcggcc 2280 tattggttaa aaaatgagctgatttaacaa aaatttaacg cgaattaatt ctgtggaatg 2340 tgtgtcagtt agggtgtggaaagtccccag gctccccagc aggcagaagt atgcaaagca 2400 tgcatctcaa ttagtcagcaaccaggtgtg gaaagtcccc aggctcccca gcaggcagaa 2460 gtatgcaaag catgcatctcaattagtcag caaccatagt cccgccccta actccgccca 2520 tcccgcccct aactccgcccagttccgccc attctccgcc ccatggctga ctaatttttt 2580 ttatttatgc agaggccgaggccgcctctg cctctgagct attccagaag tagtgaggag 2640 gcttttttgg aggcctaggcttttgcaaaa agctcccggg agcttgtata tccattttcg 2700 gatctgatca gcacgtgttgacaattaatc atcggcatag tatatcggca tagtataata 2760 cgacaaggtg aggaactaaaccatggccaa gttgaccagt gccgttccgg tgctcaccgc 2820 gcgcgacgtc gccggagcggtcgagttctg gaccgaccgg ctcgggttct cccgggactt 2880 cgtggaggac gacttcgccggtgtggtccg ggacgacgtg accctgttca tcagcgcggt 2940 ccaggaccag gtggtgccggacaacaccct ggcctgggtg tgggtgcgcg gcctggacga 3000 gctgtacgcc gagtggtcggaggtcgtgtc cacgaacttc cgggacgcct ccgggccggc 3060 catgaccgag atcggcgagcagccgtgggg gcgggagttc gccctgcgcg acccggccgg 3120 caactgcgtg cacttcgtggccgaggagca ggactgacac gtgctacgag atttcgattc 3180 caccgccgcc ttctatgaaaggttgggctt cggaatcgtt ttccgggacg ccggctggat 3240 gatcctccag cgcggggatctcatgctgga gttcttcgcc caccccaact tgtttattgc 3300 agcttataat ggttacaaataaagcaatag catcacaaat ttcacaaata aagcattttt 3360 ttcactgcat tctagttgtggtttgtccaa actcatcaat gtatcttatc atgtctgtat 3420 accgtcgacc tctagctagagcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 3480 ttgttatccg ctcacaattccacacaacat acgagccgga agcataaagt gtaaagcctg 3540 gggtgcctaa tgagtgagctaactcacatt aattgcgttg cgctcactgc ccgctttcca 3600 gtcgggaaac ctgtcgtgccagctgcatta atgaatcggc caacgcgcgg ggagaggcgg 3660 tttgcgtatt gggcgctcttccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3720 gctgcggcga gcggtatcagctcactcaaa ggcggtaata cggttatcca cagaatcagg 3780 ggataacgca ggaaagaacatgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 3840 ggccgcgttg ctggcgtttttccataggct ccgcccccct gacgagcatc acaaaaatcg 3900 acgctcaagt cagaggtggcgaaacccgac aggactataa agataccagg cgtttccccc 3960 tggaagctcc ctcgtgcgctctcctgttcc gaccctgccg cttaccggat acctgtccgc 4020 ctttctccct tcgggaagcgtggcgctttc tcaatgctca cgctgtaggt atctcagttc 4080 ggtgtaggtc gttcgctccaagctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4140 ctgcgcctta tccggtaactatcgtcttga gtccaacccg gtaagacacg acttatcgcc 4200 actggcagca gccactggtaacaggattag cagagcgagg tatgtaggcg gtgctacaga 4260 gttcttgaag tggtggcctaactacggcta cactagaagg acagtatttg gtatctgcgc 4320 tctgctgaag ccagttaccttcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4380 caccgctggt agcggtggtttttttgtttg caagcagcag attacgcgca gaaaaaaagg 4440 atctcaagaa gatcctttgatcttttctac ggggtctgac gctcagtgga acgaaaactc 4500 acgttaaggg attttggtcatgagattatc aaaaaggatc ttcacctaga tccttttaaa 4560 ttaaaaatga agttttaaatcaatctaaag tatatatgag taaacttggt ctgacagtta 4620 ccaatgctta atcagtgaggcacctatctc agcgatctgt ctatttcgtt catccatagt 4680 tgcctgactc cccgtcgtgtagataactac gatacgggag ggcttaccat ctggccccag 4740 tgctgcaatg ataccgcgagacccacgctc accggctcca gatttatcag caataaacca 4800 gccagccgga agggccgagcgcagaagtgg tcctgcaact ttatccgcct ccatccagtc 4860 tattaattgt tgccgggaagctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 4920 tgttgccatt gctacaggcatcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 4980 ctccggttcc caacgatcaaggcgagttac atgatccccc atgttgtgca aaaaagcggt 5040 tagctccttc ggtcctccgatcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5100 ggttatggca gcactgcataattctcttac tgtcatgcca tccgtaagat gcttttctgt 5160 gactggtgag tactcaaccaagtcattctg agaatagtgt atgcggcgac cgagttgctc 5220 ttgcccggcg tcaatacgggataataccgc gccacatagc agaactttaa aagtgctcat 5280 cattggaaaa cgttcttcggggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5340 ttcgatgtaa cccactcgtgcacccaactg atcttcagca tcttttactt tcaccagcgt 5400 ttctgggtga gcaaaaacaggaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5460 gaaatgttga atactcatactcttcctttt tcaatattat tgaagcattt atcagggtta 5520 ttgtctcatg agcggatacatatttgaatg tatttagaaa aataaacaaa taggggttcc 5580 gcgcacattt ccccgaaaagtgccacctga cgtc 5614 <210> SEQ ID NO 9 <211> LENGTH: 5695 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:pSecTagA-VEGF-B186-H6-NXT <400> SEQUENCE: 9 gacggatcgg gagatctcccgatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtatctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctacaacaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcgctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggactatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgaccttatgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa gctggctagt 900 ccagtgtggt ggaattcggcttcaccatga gccctctgct ccgccgcctg ctgctcgccg 960 cactcctgca gctggcccccgcccaggccc ctgtctccca gcctgatgcc cctggccacc 1020 agaggaaagt ggtgtcatggatagatgtgt atactcgcgc tacctgccag ccccgggagg 1080 tggtggtgcc cttgactgtggagctcatgg gcaccgtggc caaacagctg gtgcccagct 1140 gcgtgactgt gcagcgctgtggtggctgct gccctgacga tggcctggag tgtgtgccca 1200 ctgggcagca caacgtcaccatgcagatcc tcatgatccg gtacccgagc agtcagctgg 1260 gggagatgtc cctggaagaacacagccagt gtgaatgcag acctaaaaaa aaggacagtg 1320 ctgtgaagcc agacagggctgccactcccc accaccgtcc ccagccccgt tctgttccgg 1380 gctgggactc tgcccccggagcaccctccc cagctgacat cacccatccc actccagccc 1440 caggcccctc tgcccacgctgcacccagca ccaccagcgc cctgaccccc ggacctgccg 1500 ccgccgctgc cgacgccgcagcttcctccg ttgccaaggg cggggctcat catcatcatc 1560 atcattgaat tctgcagatatccagcacag tggcggccgc tcgagtctag agggcccgaa 1620 caaaaactca tctcagaagaggatctgaat agcgccgtcg accatcatca tcatcatcat 1680 tgagtttaaa cccgctgatcagcctcgact gtgccttcta gttgccagcc atctgttgtt 1740 tgcccctccc ccgtgccttccttgaccctg gaaggtgcca ctcccactgt cctttcctaa 1800 taaaatgagg aaattgcatcgcattgtctg agtaggtgtc attctattct ggggggtggg 1860 gtggggcagg acagcaagggggaggattgg gaagacaata gcaggcatgc tggggatgcg 1920 gtgggctcta tggcttctgaggcggaaaga accagctggg gctctagggg gtatccccac 1980 gcgccctgta gcggcgcattaagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 2040 acacttgcca gcgccctagcgcccgctcct ttcgctttct tcccttcctt tctcgccacg 2100 ttcgccggct ttccccgtcaagctctaaat cggggcatcc ctttagggtt ccgatttagt 2160 gctttacggc acctcgaccccaaaaaactt gattagggtg atggttcacg tagtgggcca 2220 tcgccctgat agacggtttttcgccctttg acgttggagt ccacgttctt taatagtgga 2280 ctcttgttcc aaactggaacaacactcaac cctatctcgg tctattcttt tgatttataa 2340 gggattttgg ggatttcggcctattggtta aaaaatgagc tgatttaaca aaaatttaac 2400 gcgaattaat tctgtggaatgtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 2460 caggcagaag tatgcaaagcatgcatctca attagtcagc aaccaggtgt ggaaagtccc 2520 caggctcccc agcaggcagaagtatgcaaa gcatgcatct caattagtca gcaaccatag 2580 tcccgcccct aactccgcccatcccgcccc taactccgcc cagttccgcc cattctccgc 2640 cccatggctg actaattttttttatttatg cagaggccga ggccgcctct gcctctgagc 2700 tattccagaa gtagtgaggaggcttttttg gaggcctagg cttttgcaaa aagctcccgg 2760 gagcttgtat atccattttcggatctgatc agcacgtgtt gacaattaat catcggcata 2820 gtatatcggc atagtataatacgacaaggt gaggaactaa accatggcca agttgaccag 2880 tgccgttccg gtgctcaccgcgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg 2940 gctcgggttc tcccgggacttcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt 3000 gaccctgttc atcagcgcggtccaggacca ggtggtgccg gacaacaccc tggcctgggt 3060 gtgggtgcgc ggcctggacgagctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt 3120 ccgggacgcc tccgggccggccatgaccga gatcggcgag cagccgtggg ggcgggagtt 3180 cgccctgcgc gacccggccggcaactgcgt gcacttcgtg gccgaggagc aggactgaca 3240 cgtgctacga gatttcgattccaccgccgc cttctatgaa aggttgggct tcggaatcgt 3300 tttccgggac gccggctggatgatcctcca gcgcggggat ctcatgctgg agttcttcgc 3360 ccaccccaac ttgtttattgcagcttataa tggttacaaa taaagcaata gcatcacaaa 3420 tttcacaaat aaagcatttttttcactgca ttctagttgt ggtttgtcca aactcatcaa 3480 tgtatcttat catgtctgtataccgtcgac ctctagctag agcttggcgt aatcatggtc 3540 atagctgttt cctgtgtgaaattgttatcc gctcacaatt ccacacaaca tacgagccgg 3600 aagcataaag tgtaaagcctggggtgccta atgagtgagc taactcacat taattgcgtt 3660 gcgctcactg cccgctttccagtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3720 ccaacgcgcg gggagaggcggtttgcgtat tgggcgctct tccgcttcct cgctcactga 3780 ctcgctgcgc tcggtcgttcggctgcggcg agcggtatca gctcactcaa aggcggtaat 3840 acggttatcc acagaatcaggggataacgc aggaaagaac atgtgagcaa aaggccagca 3900 aaaggccagg aaccgtaaaaaggccgcgtt gctggcgttt ttccataggc tccgcccccc 3960 tgacgagcat cacaaaaatcgacgctcaag tcagaggtgg cgaaacccga caggactata 4020 aagataccag gcgtttccccctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4080 gcttaccgga tacctgtccgcctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4140 acgctgtagg tatctcagttcggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4200 accccccgtt cagcccgaccgctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4260 ggtaagacac gacttatcgccactggcagc agccactggt aacaggatta gcagagcgag 4320 gtatgtaggc ggtgctacagagttcttgaa gtggtggcct aactacggct acactagaag 4380 gacagtattt ggtatctgcgctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4440 ctcttgatcc ggcaaacaaaccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4500 gattacgcgc agaaaaaaaggatctcaaga agatcctttg atcttttcta cggggtctga 4560 cgctcagtgg aacgaaaactcacgttaagg gattttggtc atgagattat caaaaaggat 4620 cttcacctag atccttttaaattaaaaatg aagttttaaa tcaatctaaa gtatatatga 4680 gtaaacttgg tctgacagttaccaatgctt aatcagtgag gcacctatct cagcgatctg 4740 tctatttcgt tcatccatagttgcctgact ccccgtcgtg tagataacta cgatacggga 4800 gggcttacca tctggccccagtgctgcaat gataccgcga gacccacgct caccggctcc 4860 agatttatca gcaataaaccagccagccgg aagggccgag cgcagaagtg gtcctgcaac 4920 tttatccgcc tccatccagtctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4980 agttaatagt ttgcgcaacgttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 5040 gtttggtatg gcttcattcagctccggttc ccaacgatca aggcgagtta catgatcccc 5100 catgttgtgc aaaaaagcggttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5160 ggccgcagtg ttatcactcatggttatggc agcactgcat aattctctta ctgtcatgcc 5220 atccgtaaga tgcttttctgtgactggtga gtactcaacc aagtcattct gagaatagtg 5280 tatgcggcga ccgagttgctcttgcccggc gtcaatacgg gataataccg cgccacatag 5340 cagaacttta aaagtgctcatcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5400 cttaccgctg ttgagatccagttcgatgta acccactcgt gcacccaact gatcttcagc 5460 atcttttact ttcaccagcgtttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5520 aaagggaata agggcgacacggaaatgttg aatactcata ctcttccttt ttcaatatta 5580 ttgaagcatt tatcagggttattgtctcat gagcggatac atatttgaat gtatttagaa 5640 aaataaacaa ataggggttccgcgcacatt tccccgaaaa gtgccacctg acgtc 5695 <210> SEQ ID NO 10 <211>LENGTH: 5695 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:pSecTagA-VEGF-B186-H6 <400> SEQUENCE: 10 gacggatcgg gagatctcccgatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtatctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctacaacaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcgctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggactatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgaccttatgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa gctggctagt 900 ccagtgtggt ggaattcggcttcaccatga gccctctgct ccgccgcctg ctgctcgccg 960 cactcctgca gctggcccccgcccaggccc ctgtctccca gcctgatgcc cctggccacc 1020 agaggaaagt ggtgtcatggatagatgtgt atactcgcgc tacctgccag ccccgggagg 1080 tggtggtgcc cttgactgtggagctcatgg gcaccgtggc caaacagctg gtgcccagct 1140 gcgtgactgt gcagcgctgtggtggctgct gccctgacga tggcctggag tgtgtgccca 1200 ctgggcagca ccaagtccggatgcagatcc tcatgatccg gtacccgagc agtcagctgg 1260 gggagatgtc cctggaagaacacagccagt gtgaatgcag acctaaaaaa aaggacagtg 1320 ctgtgaagcc agacagggctgccactcccc accaccgtcc ccagccccgt tctgttccgg 1380 gctgggactc tgcccccggagcaccctccc cagctgacat cacccatccc actccagccc 1440 caggcccctc tgcccacgctgcacccagca ccaccagcgc cctgaccccc ggacctgccg 1500 ccgccgctgc cgacgccgcagcttcctccg ttgccaaggg cggggctcat catcatcatc 1560 atcattgaat tctgcagatatccagcacag tggcggccgc tcgagtctag agggcccgaa 1620 caaaaactca tctcagaagaggatctgaat agcgccgtcg accatcatca tcatcatcat 1680 tgagtttaaa cccgctgatcagcctcgact gtgccttcta gttgccagcc atctgttgtt 1740 tgcccctccc ccgtgccttccttgaccctg gaaggtgcca ctcccactgt cctttcctaa 1800 taaaatgagg aaattgcatcgcattgtctg agtaggtgtc attctattct ggggggtggg 1860 gtggggcagg acagcaagggggaggattgg gaagacaata gcaggcatgc tggggatgcg 1920 gtgggctcta tggcttctgaggcggaaaga accagctggg gctctagggg gtatccccac 1980 gcgccctgta gcggcgcattaagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 2040 acacttgcca gcgccctagcgcccgctcct ttcgctttct tcccttcctt tctcgccacg 2100 ttcgccggct ttccccgtcaagctctaaat cggggcatcc ctttagggtt ccgatttagt 2160 gctttacggc acctcgaccccaaaaaactt gattagggtg atggttcacg tagtgggcca 2220 tcgccctgat agacggtttttcgccctttg acgttggagt ccacgttctt taatagtgga 2280 ctcttgttcc aaactggaacaacactcaac cctatctcgg tctattcttt tgatttataa 2340 gggattttgg ggatttcggcctattggtta aaaaatgagc tgatttaaca aaaatttaac 2400 gcgaattaat tctgtggaatgtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 2460 caggcagaag tatgcaaagcatgcatctca attagtcagc aaccaggtgt ggaaagtccc 2520 caggctcccc agcaggcagaagtatgcaaa gcatgcatct caattagtca gcaaccatag 2580 tcccgcccct aactccgcccatcccgcccc taactccgcc cagttccgcc cattctccgc 2640 cccatggctg actaattttttttatttatg cagaggccga ggccgcctct gcctctgagc 2700 tattccagaa gtagtgaggaggcttttttg gaggcctagg cttttgcaaa aagctcccgg 2760 gagcttgtat atccattttcggatctgatc agcacgtgtt gacaattaat catcggcata 2820 gtatatcggc atagtataatacgacaaggt gaggaactaa accatggcca agttgaccag 2880 tgccgttccg gtgctcaccgcgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg 2940 gctcgggttc tcccgggacttcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt 3000 gaccctgttc atcagcgcggtccaggacca ggtggtgccg gacaacaccc tggcctgggt 3060 gtgggtgcgc ggcctggacgagctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt 3120 ccgggacgcc tccgggccggccatgaccga gatcggcgag cagccgtggg ggcgggagtt 3180 cgccctgcgc gacccggccggcaactgcgt gcacttcgtg gccgaggagc aggactgaca 3240 cgtgctacga gatttcgattccaccgccgc cttctatgaa aggttgggct tcggaatcgt 3300 tttccgggac gccggctggatgatcctcca gcgcggggat ctcatgctgg agttcttcgc 3360 ccaccccaac ttgtttattgcagcttataa tggttacaaa taaagcaata gcatcacaaa 3420 tttcacaaat aaagcatttttttcactgca ttctagttgt ggtttgtcca aactcatcaa 3480 tgtatcttat catgtctgtataccgtcgac ctctagctag agcttggcgt aatcatggtc 3540 atagctgttt cctgtgtgaaattgttatcc gctcacaatt ccacacaaca tacgagccgg 3600 aagcataaag tgtaaagcctggggtgccta atgagtgagc taactcacat taattgcgtt 3660 gcgctcactg cccgctttccagtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3720 ccaacgcgcg gggagaggcggtttgcgtat tgggcgctct tccgcttcct cgctcactga 3780 ctcgctgcgc tcggtcgttcggctgcggcg agcggtatca gctcactcaa aggcggtaat 3840 acggttatcc acagaatcaggggataacgc aggaaagaac atgtgagcaa aaggccagca 3900 aaaggccagg aaccgtaaaaaggccgcgtt gctggcgttt ttccataggc tccgcccccc 3960 tgacgagcat cacaaaaatcgacgctcaag tcagaggtgg cgaaacccga caggactata 4020 aagataccag gcgtttccccctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4080 gcttaccgga tacctgtccgcctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4140 acgctgtagg tatctcagttcggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4200 accccccgtt cagcccgaccgctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4260 ggtaagacac gacttatcgccactggcagc agccactggt aacaggatta gcagagcgag 4320 gtatgtaggc ggtgctacagagttcttgaa gtggtggcct aactacggct acactagaag 4380 gacagtattt ggtatctgcgctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4440 ctcttgatcc ggcaaacaaaccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4500 gattacgcgc agaaaaaaaggatctcaaga agatcctttg atcttttcta cggggtctga 4560 cgctcagtgg aacgaaaactcacgttaagg gattttggtc atgagattat caaaaaggat 4620 cttcacctag atccttttaaattaaaaatg aagttttaaa tcaatctaaa gtatatatga 4680 gtaaacttgg tctgacagttaccaatgctt aatcagtgag gcacctatct cagcgatctg 4740 tctatttcgt tcatccatagttgcctgact ccccgtcgtg tagataacta cgatacggga 4800 gggcttacca tctggccccagtgctgcaat gataccgcga gacccacgct caccggctcc 4860 agatttatca gcaataaaccagccagccgg aagggccgag cgcagaagtg gtcctgcaac 4920 tttatccgcc tccatccagtctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4980 agttaatagt ttgcgcaacgttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 5040 gtttggtatg gcttcattcagctccggttc ccaacgatca aggcgagtta catgatcccc 5100 catgttgtgc aaaaaagcggttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5160 ggccgcagtg ttatcactcatggttatggc agcactgcat aattctctta ctgtcatgcc 5220 atccgtaaga tgcttttctgtgactggtga gtactcaacc aagtcattct gagaatagtg 5280 tatgcggcga ccgagttgctcttgcccggc gtcaatacgg gataataccg cgccacatag 5340 cagaacttta aaagtgctcatcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5400 cttaccgctg ttgagatccagttcgatgta acccactcgt gcacccaact gatcttcagc 5460 atcttttact ttcaccagcgtttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5520 aaagggaata agggcgacacggaaatgttg aatactcata ctcttccttt ttcaatatta 5580 ttgaagcatt tatcagggttattgtctcat gagcggatac atatttgaat gtatttagaa 5640 aaataaacaa ataggggttccgcgcacatt tccccgaaaa gtgccacctg acgtc 5695 <210> SEQ ID NO 11 <211>LENGTH: 5458 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:pSecTagA-VEGF-BEx1-5-H6 <400> SEQUENCE: 11 gacggatcgg gagatctcccgatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtatctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctacaacaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcgctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggactatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgaccttatgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa gctggctagt 900 ccagtgtggt ggaattcggcttcaccatga gccctctgct ccgccgcctg ctgctcgccg 960 cactcctgca gctggcccccgcccaggccc ctgtctccca gcctgatgcc cctggccacc 1020 agaggaaagt ggtgtcatggatagatgtgt atactcgcgc tacctgccag ccccgggagg 1080 tggtggtgcc cttgactgtggagctcatgg gcaccgtggc caaacagctg gtgcccagct 1140 gcgtgactgt gcagcgctgtggtggctgct gccctgacga tggcctggag tgtgtgccca 1200 ctgggcagca ccaagtccggatgcagatcc tcatgatccg gtacccgagc agtcagctgg 1260 gggagatgtc cctggaagaacacagccagt gtgaatgcag acctaaaaaa aaggacagtg 1320 ctgtgaagcc agaccatcatcatcatcacc actgagcggc cgctcgagtc tagagggccc 1380 gaacaaaaac tcatctcagaagaggatctg aatagcgccg tcgaccatca tcatcatcat 1440 cattgagttt aaacccgctgatcagcctcg actgtgcctt ctagttgcca gccatctgtt 1500 gtttgcccct cccccgtgccttccttgacc ctggaaggtg ccactcccac tgtcctttcc 1560 taataaaatg aggaaattgcatcgcattgt ctgagtaggt gtcattctat tctggggggt 1620 ggggtggggc aggacagcaagggggaggat tgggaagaca atagcaggca tgctggggat 1680 gcggtgggct ctatggcttctgaggcggaa agaaccagct ggggctctag ggggtatccc 1740 cacgcgccct gtagcggcgcattaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 1800 gctacacttg ccagcgccctagcgcccgct cctttcgctt tcttcccttc ctttctcgcc 1860 acgttcgccg gctttccccgtcaagctcta aatcggggca tccctttagg gttccgattt 1920 agtgctttac ggcacctcgaccccaaaaaa cttgattagg gtgatggttc acgtagtggg 1980 ccatcgccct gatagacggtttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2040 ggactcttgt tccaaactggaacaacactc aaccctatct cggtctattc ttttgattta 2100 taagggattt tggggatttcggcctattgg ttaaaaaatg agctgattta acaaaaattt 2160 aacgcgaatt aattctgtggaatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 2220 cagcaggcag aagtatgcaaagcatgcatc tcaattagtc agcaaccagg tgtggaaagt 2280 ccccaggctc cccagcaggcagaagtatgc aaagcatgca tctcaattag tcagcaacca 2340 tagtcccgcc cctaactccgcccatcccgc ccctaactcc gcccagttcc gcccattctc 2400 cgccccatgg ctgactaattttttttattt atgcagaggc cgaggccgcc tctgcctctg 2460 agctattcca gaagtagtgaggaggctttt ttggaggcct aggcttttgc aaaaagctcc 2520 cgggagcttg tatatccattttcggatctg atcagcacgt gttgacaatt aatcatcggc 2580 atagtatatc ggcatagtataatacgacaa ggtgaggaac taaaccatgg ccaagttgac 2640 cagtgccgtt ccggtgctcaccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga 2700 ccggctcggg ttctcccgggacttcgtgga ggacgacttc gccggtgtgg tccgggacga 2760 cgtgaccctg ttcatcagcgcggtccagga ccaggtggtg ccggacaaca ccctggcctg 2820 ggtgtgggtg cgcggcctggacgagctgta cgccgagtgg tcggaggtcg tgtccacgaa 2880 cttccgggac gcctccgggccggccatgac cgagatcggc gagcagccgt gggggcggga 2940 gttcgccctg cgcgacccggccggcaactg cgtgcacttc gtggccgagg agcaggactg 3000 acacgtgcta cgagatttcgattccaccgc cgccttctat gaaaggttgg gcttcggaat 3060 cgttttccgg gacgccggctggatgatcct ccagcgcggg gatctcatgc tggagttctt 3120 cgcccacccc aacttgtttattgcagctta taatggttac aaataaagca atagcatcac 3180 aaatttcaca aataaagcatttttttcact gcattctagt tgtggtttgt ccaaactcat 3240 caatgtatct tatcatgtctgtataccgtc gacctctagc tagagcttgg cgtaatcatg 3300 gtcatagctg tttcctgtgtgaaattgtta tccgctcaca attccacaca acatacgagc 3360 cggaagcata aagtgtaaagcctggggtgc ctaatgagtg agctaactca cattaattgc 3420 gttgcgctca ctgcccgctttccagtcggg aaacctgtcg tgccagctgc attaatgaat 3480 cggccaacgc gcggggagaggcggtttgcg tattgggcgc tcttccgctt cctcgctcac 3540 tgactcgctg cgctcggtcgttcggctgcg gcgagcggta tcagctcact caaaggcggt 3600 aatacggtta tccacagaatcaggggataa cgcaggaaag aacatgtgag caaaaggcca 3660 gcaaaaggcc aggaaccgtaaaaaggccgc gttgctggcg tttttccata ggctccgccc 3720 ccctgacgag catcacaaaaatcgacgctc aagtcagagg tggcgaaacc cgacaggact 3780 ataaagatac caggcgtttccccctggaag ctccctcgtg cgctctcctg ttccgaccct 3840 gccgcttacc ggatacctgtccgcctttct cccttcggga agcgtggcgc tttctcaatg 3900 ctcacgctgt aggtatctcagttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 3960 cgaacccccc gttcagcccgaccgctgcgc cttatccggt aactatcgtc ttgagtccaa 4020 cccggtaaga cacgacttatcgccactggc agcagccact ggtaacagga ttagcagagc 4080 gaggtatgta ggcggtgctacagagttctt gaagtggtgg cctaactacg gctacactag 4140 aaggacagta tttggtatctgcgctctgct gaagccagtt accttcggaa aaagagttgg 4200 tagctcttga tccggcaaacaaaccaccgc tggtagcggt ggtttttttg tttgcaagca 4260 gcagattacg cgcagaaaaaaaggatctca agaagatcct ttgatctttt ctacggggtc 4320 tgacgctcag tggaacgaaaactcacgtta agggattttg gtcatgagat tatcaaaaag 4380 gatcttcacc tagatccttttaaattaaaa atgaagtttt aaatcaatct aaagtatata 4440 tgagtaaact tggtctgacagttaccaatg cttaatcagt gaggcaccta tctcagcgat 4500 ctgtctattt cgttcatccatagttgcctg actccccgtc gtgtagataa ctacgatacg 4560 ggagggctta ccatctggccccagtgctgc aatgataccg cgagacccac gctcaccggc 4620 tccagattta tcagcaataaaccagccagc cggaagggcc gagcgcagaa gtggtcctgc 4680 aactttatcc gcctccatccagtctattaa ttgttgccgg gaagctagag taagtagttc 4740 gccagttaat agtttgcgcaacgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 4800 gtcgtttggt atggcttcattcagctccgg ttcccaacga tcaaggcgag ttacatgatc 4860 ccccatgttg tgcaaaaaagcggttagctc cttcggtcct ccgatcgttg tcagaagtaa 4920 gttggccgca gtgttatcactcatggttat ggcagcactg cataattctc ttactgtcat 4980 gccatccgta agatgcttttctgtgactgg tgagtactca accaagtcat tctgagaata 5040 gtgtatgcgg cgaccgagttgctcttgccc ggcgtcaata cgggataata ccgcgccaca 5100 tagcagaact ttaaaagtgctcatcattgg aaaacgttct tcggggcgaa aactctcaag 5160 gatcttaccg ctgttgagatccagttcgat gtaacccact cgtgcaccca actgatcttc 5220 agcatctttt actttcaccagcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 5280 aaaaaaggga ataagggcgacacggaaatg ttgaatactc atactcttcc tttttcaata 5340 ttattgaagc atttatcagggttattgtct catgagcgga tacatatttg aatgtattta 5400 gaaaaataaa caaataggggttccgcgcac atttccccga aaagtgccac ctgacgtc 5458 <210> SEQ ID NO 12 <211>LENGTH: 5458 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:pSecTagA-VEGF-BEx1-5-H6-NXT <400> SEQUENCE: 12 gacggatcgg gagatctcccgatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtatctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctacaacaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcgctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggactatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgaccttatgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa gctggctagt 900 ccagtgtggt ggaattcggcttcaccatga gccctctgct ccgccgcctg ctgctcgccg 960 cactcctgca gctggcccccgcccaggccc ctgtctccca gcctgatgcc cctggccacc 1020 agaggaaagt ggtgtcatggatagatgtgt atactcgcgc tacctgccag ccccgggagg 1080 tggtggtgcc cttgactgtggagctcatgg gcaccgtggc caaacagctg gtgcccagct 1140 gcgtgactgt gcagcgctgtggtggctgct gccctgacga tggcctggag tgtgtgccca 1200 ctgggcagca caacgtcaccatgcagatcc tcatgatccg gtacccgagc agtcagctgg 1260 gggagatgtc cctggaagaacacagccagt gtgaatgcag acctaaaaaa aaggacagtg 1320 ctgtgaagcc agaccatcatcatcatcacc actgagcggc cgctcgagtc tagagggccc 1380 gaacaaaaac tcatctcagaagaggatctg aatagcgccg tcgaccatca tcatcatcat 1440 cattgagttt aaacccgctgatcagcctcg actgtgcctt ctagttgcca gccatctgtt 1500 gtttgcccct cccccgtgccttccttgacc ctggaaggtg ccactcccac tgtcctttcc 1560 taataaaatg aggaaattgcatcgcattgt ctgagtaggt gtcattctat tctggggggt 1620 ggggtggggc aggacagcaagggggaggat tgggaagaca atagcaggca tgctggggat 1680 gcggtgggct ctatggcttctgaggcggaa agaaccagct ggggctctag ggggtatccc 1740 cacgcgccct gtagcggcgcattaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 1800 gctacacttg ccagcgccctagcgcccgct cctttcgctt tcttcccttc ctttctcgcc 1860 acgttcgccg gctttccccgtcaagctcta aatcggggca tccctttagg gttccgattt 1920 agtgctttac ggcacctcgaccccaaaaaa cttgattagg gtgatggttc acgtagtggg 1980 ccatcgccct gatagacggtttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2040 ggactcttgt tccaaactggaacaacactc aaccctatct cggtctattc ttttgattta 2100 taagggattt tggggatttcggcctattgg ttaaaaaatg agctgattta acaaaaattt 2160 aacgcgaatt aattctgtggaatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 2220 cagcaggcag aagtatgcaaagcatgcatc tcaattagtc agcaaccagg tgtggaaagt 2280 ccccaggctc cccagcaggcagaagtatgc aaagcatgca tctcaattag tcagcaacca 2340 tagtcccgcc cctaactccgcccatcccgc ccctaactcc gcccagttcc gcccattctc 2400 cgccccatgg ctgactaattttttttattt atgcagaggc cgaggccgcc tctgcctctg 2460 agctattcca gaagtagtgaggaggctttt ttggaggcct aggcttttgc aaaaagctcc 2520 cgggagcttg tatatccattttcggatctg atcagcacgt gttgacaatt aatcatcggc 2580 atagtatatc ggcatagtataatacgacaa ggtgaggaac taaaccatgg ccaagttgac 2640 cagtgccgtt ccggtgctcaccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga 2700 ccggctcggg ttctcccgggacttcgtgga ggacgacttc gccggtgtgg tccgggacga 2760 cgtgaccctg ttcatcagcgcggtccagga ccaggtggtg ccggacaaca ccctggcctg 2820 ggtgtgggtg cgcggcctggacgagctgta cgccgagtgg tcggaggtcg tgtccacgaa 2880 cttccgggac gcctccgggccggccatgac cgagatcggc gagcagccgt gggggcggga 2940 gttcgccctg cgcgacccggccggcaactg cgtgcacttc gtggccgagg agcaggactg 3000 acacgtgcta cgagatttcgattccaccgc cgccttctat gaaaggttgg gcttcggaat 3060 cgttttccgg gacgccggctggatgatcct ccagcgcggg gatctcatgc tggagttctt 3120 cgcccacccc aacttgtttattgcagctta taatggttac aaataaagca atagcatcac 3180 aaatttcaca aataaagcatttttttcact gcattctagt tgtggtttgt ccaaactcat 3240 caatgtatct tatcatgtctgtataccgtc gacctctagc tagagcttgg cgtaatcatg 3300 gtcatagctg tttcctgtgtgaaattgtta tccgctcaca attccacaca acatacgagc 3360 cggaagcata aagtgtaaagcctggggtgc ctaatgagtg agctaactca cattaattgc 3420 gttgcgctca ctgcccgctttccagtcggg aaacctgtcg tgccagctgc attaatgaat 3480 cggccaacgc gcggggagaggcggtttgcg tattgggcgc tcttccgctt cctcgctcac 3540 tgactcgctg cgctcggtcgttcggctgcg gcgagcggta tcagctcact caaaggcggt 3600 aatacggtta tccacagaatcaggggataa cgcaggaaag aacatgtgag caaaaggcca 3660 gcaaaaggcc aggaaccgtaaaaaggccgc gttgctggcg tttttccata ggctccgccc 3720 ccctgacgag catcacaaaaatcgacgctc aagtcagagg tggcgaaacc cgacaggact 3780 ataaagatac caggcgtttccccctggaag ctccctcgtg cgctctcctg ttccgaccct 3840 gccgcttacc ggatacctgtccgcctttct cccttcggga agcgtggcgc tttctcaatg 3900 ctcacgctgt aggtatctcagttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 3960 cgaacccccc gttcagcccgaccgctgcgc cttatccggt aactatcgtc ttgagtccaa 4020 cccggtaaga cacgacttatcgccactggc agcagccact ggtaacagga ttagcagagc 4080 gaggtatgta ggcggtgctacagagttctt gaagtggtgg cctaactacg gctacactag 4140 aaggacagta tttggtatctgcgctctgct gaagccagtt accttcggaa aaagagttgg 4200 tagctcttga tccggcaaacaaaccaccgc tggtagcggt ggtttttttg tttgcaagca 4260 gcagattacg cgcagaaaaaaaggatctca agaagatcct ttgatctttt ctacggggtc 4320 tgacgctcag tggaacgaaaactcacgtta agggattttg gtcatgagat tatcaaaaag 4380 gatcttcacc tagatccttttaaattaaaa atgaagtttt aaatcaatct aaagtatata 4440 tgagtaaact tggtctgacagttaccaatg cttaatcagt gaggcaccta tctcagcgat 4500 ctgtctattt cgttcatccatagttgcctg actccccgtc gtgtagataa ctacgatacg 4560 ggagggctta ccatctggccccagtgctgc aatgataccg cgagacccac gctcaccggc 4620 tccagattta tcagcaataaaccagccagc cggaagggcc gagcgcagaa gtggtcctgc 4680 aactttatcc gcctccatccagtctattaa ttgttgccgg gaagctagag taagtagttc 4740 gccagttaat agtttgcgcaacgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 4800 gtcgtttggt atggcttcattcagctccgg ttcccaacga tcaaggcgag ttacatgatc 4860 ccccatgttg tgcaaaaaagcggttagctc cttcggtcct ccgatcgttg tcagaagtaa 4920 gttggccgca gtgttatcactcatggttat ggcagcactg cataattctc ttactgtcat 4980 gccatccgta agatgcttttctgtgactgg tgagtactca accaagtcat tctgagaata 5040 gtgtatgcgg cgaccgagttgctcttgccc ggcgtcaata cgggataata ccgcgccaca 5100 tagcagaact ttaaaagtgctcatcattgg aaaacgttct tcggggcgaa aactctcaag 5160 gatcttaccg ctgttgagatccagttcgat gtaacccact cgtgcaccca actgatcttc 5220 agcatctttt actttcaccagcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 5280 aaaaaaggga ataagggcgacacggaaatg ttgaatactc atactcttcc tttttcaata 5340 ttattgaagc atttatcagggttattgtct catgagcgga tacatatttg aatgtattta 5400 gaaaaataaa caaataggggttccgcgcac atttccccga aaagtgccac ctgacgtc 5458 <210> SEQ ID NO 13 <211>LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR primer to introduce a N-glycosylation site at positions 289-297 ofSEQ ID NO:3 (VEGF-B186) <400> SEQUENCE: 13 tcggtaccgg atcatgaggatctgcatggt gacgttgtgc tgcccagtgg cca 53 <210> SEQ ID NO 14 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: PCR 5′primer for amplification of nucleotides 250 to 567 from Genebank Acc.No. U48801 <400> SEQUENCE: 14 cctgacgatg gcctggagtg t 21 <210> SEQ ID NO15 <211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: PCR 3′ primer for amplification of nucleotides 250 to 567 fromGenebank Acc. No. U48801 <400> SEQUENCE: 15 gagcggccgc tcaatgatgatgatgatgat gccttcgcag cttccggcac 50 <210> SEQ ID NO 16 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: PCR 5′ primer foramplification of nucleotides 1 to 411 from Genebank Acc. No. U48801<400> SEQUENCE: 16 caccatgagc cctctgctcc 20 <210> SEQ ID NO 17 <211>LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR 3′ primer for amplification of nucleotides 1 to 411 from GenebankAcc. No. U48801 <400> SEQUENCE: 17 gagcggccgc tcagtggtga tgatgatggtctggcttcac agcactg 47

What is claimed is:
 1. An isolated nucleic acid molecule comprising: a polynucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or a polynucleotide sequence which hybridizes under stringent conditions with at least one of the foregoing sequences; and a nucleotide sequence encoding at least one putative N-glycosylation site inserted therein.
 2. An isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 and having at least one putative N-glycosylation site inserted therein.
 3. The isolated nucleic acid molecule of claim 1, wherein the at least one putative N-glycosylation site consists of a nucleotide sequence that encodes an amino acid sequence of NXT.
 4. The isolated nucleic acid molecule of claim 1, wherein the at least one putative N-glycosylation site is inserted at nucleotides 286-294 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
 5. An isolated polypeptide produced by expression of the nucleic acid molecule of claim
 1. 6. An isolated polypeptide of claim 2 which binds a Vascular Endothelial Growth Factor Receptor-1.
 7. A vector comprising a nucleic acid molecule of claim
 1. 8. A host cell transformed or transfected with a vector according to claim
 7. 9. A pharmaceutical composition comprising an effective amount of a polypeptide of claim
 2. 10. The pharmaceutical composition of claim 9, further comprising heparin.
 11. A method of making a soluble VEGF-B₁₆₇ from a host cell, comprising: inserting at least one putative N-glycosylation site into a nucleotide sequence of SEQ ID NO:1; transforming or transfecting said nucleotide sequence with inserted N-glycosylation site into a host cell; culturing the transfected host cell in a growth medium such that said nucleotide sequence with inserted N-glycosylation site is expressed; and isolating the expressed polypeptide from the growth medium in which said host cell was cultured.
 12. The method of claim 11, further comprising exposing the cultured transfected host cell to heparin after said polypeptide is expressed.
 13. The method of claim 11, wherein the at least one putative N-glycosylation site consists of a nucleotide sequence that encodes an amino acid sequence of NXT.
 14. The method of claim 11, wherein the nucleotide sequence encoding the at least one putative N-glycosylation site is inserted at nucleotides 286-294 of SEQ ID NO:1.
 15. A method of increasing an amount of a soluble VEGF-B₁₆₇, VEGF-B₁₈₆ or VEGF-B_(Ex1-5) polypeptide from a host cell, comprising: inserting at least one putative N-glycosylation site into a nucleotide sequence selected from the group of nucleotides sequences of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; transforming or transfecting said nucleotide sequence with inserted N-glycosylation site into a host cell; culturing the transfected host cell in a growth medium such that said nucleotide sequence with inserted N-glycosylation site is expressed; and isolating the expressed polypeptide from the growth medium in which said host cell was cultured.
 16. The method of claim 15, further comprising exposing the cultured transfected host cell to heparin after said polypeptide is expressed.
 17. The method of claim 15, wherein the at least one putative N-glycosylation site consists of a nucleotide sequence that encodes an amino acid sequence of NXT.
 18. The method of claim 15, wherein the nucleotide sequence encoding the at least one putative N-glycosylation site is inserted at nucleotides 286-294 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. 